Albumin variants

ABSTRACT

The present invention relates to variants of a parent albumin, the variants having altered plasma half-life compared with the parent albumin. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority or the benefit under 35 U.S.C. 119 ofEuropean application no. 12191856.9 filed Nov. 8, 2012 and U.S.provisional application No. 61/724,669 filed Nov. 9, 2012, the contentsof which are fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to variants of albumin or fragments thereof orfusion polypeptides comprising variant albumin or fragments thereofhaving a change in binding affinity to FcRn and/or a change in half-lifecompared to the albumin, fragment thereof or fusion polypeptidecomprising albumin or a fragment thereof. The invention allows tailoringof binding affinity and/or half-life of an albumin to the requirementsand desires of a user or application.

2. Description of the Related Art

Albumin is a protein naturally found in the blood plasma of mammalswhere it is the most abundant protein. It has important roles inmaintaining the desired osmotic pressure of the blood and also intransport of various substances in the blood stream. Albumins have beencharacterized from many species including human, pig, mouse, rat, rabbitand goat and they share a high degree of sequence and structuralhomology.

Albumin binds in vivo to its receptor, the neonatal Fc receptor (FcRn)“Brambell” and this interaction is known to be important for the plasmahalf-life of albumin. FcRn is a membrane bound protein, expressed inmany cell and tissue types. FcRn has been found to salvage albumin fromintracellular degradation (Roopenian D. C. and Akilesh, S. (2007), Nat.Rev. Immunol 7, 715-725.). FcRn is a bifunctional molecule thatcontributes to maintaining a high level of IgGs and albumin in serum inmammals such as human beings.

Whilst the FcRn-immunoglobulin (IgG) interaction has been characterizedin the prior art, the FcRn-albumin interaction is less wellcharacterized. The major FcRn binding site is localized within DIII(381-585), (Andersen et al (2010), Clinical Biochemistry 43, 367-372). Anumber of key amino acids have been shown to be important in binding,notably histidines H464, H510 and H536 and Lys500 (Andersen et al(2010), Nat. Commun. 3:610. DOI:10.1038/ncomms1607). The crystalstructure of a human serum albumin (HSA) variant(V418M+T420A+E505G+V547A) with strong affinity to FcRn at acidic pH andin addition with increased binding at neutral pH has been reportedallowing more detailed understanding of the interacting interfaces. Inaddition, the authors were able to alter the affinity to FcRn throughamino acid substitution and show that this could translate intoincreased circulatory half-lives in mice and monkey, most notably forHSA E505G+V547A (Schmidt et al (2012), Cell Structure. 21, Issue 11,(doi:10.1016/j.str.2013.08.022)).

Data indicates that IgG and albumin bind non-cooperatively to distinctsites on FcRn (Andersen et al. (2006), Eur. J. Immunol. 36, 3044-3051;Chaudhury et al. (2006), Biochemistry 45, 4983-4990).

It is known that mouse FcRn binds IgG from mice and humans whereas humanFcRn appears to be more discriminating (Ober et al. (2001) Int. Immunol13, 1551-1559). Andersen et al. (2010) Journal of Biological Chemistry285(7):4826-36, describes the affinity of human and mouse FcRn for eachmouse and human albumin (all possible combinations). No binding ofalbumin from either species was observed at physiological pH to eitherreceptor. At acidic pH, a 100-fold difference in binding affinity wasobserved. In all cases, binding of albumin and IgG from either speciesto both receptors were additive.

Human serum albumin (HSA) has been well characterized as a polypeptideof 585 amino acids, the sequence of which can be found in Peters, T.,Jr. (1996) All about Albumin: Biochemistry, Genetics and Medical,Applications pp 10, Academic Press, Inc., Orlando (ISBN 0-12-552110-3).It has a characteristic binding to its receptor FcRn, where it binds atpH 6.0 but not at pH 7.4.

The plasma half-life of HSA has been found to be approximately 19 days.A natural variant having lower plasma half-life has been identified(Peach, R. J. and Brennan, S. 0., (1991) Biochim Biophys Acta.1097:49-54) having the substitution D494N. This substitution generatedan N-glycosylation site in this variant, which is not present in thewild-type albumin. It is not known whether the glycosylation or theamino acid change is responsible for the change in plasma half-life.

Albumin has a long plasma half-life and because of this property it hasbeen suggested for use in drug delivery. Albumin has been conjugated topharmaceutically beneficial compounds (WO2000/69902), and it was foundthat the conjugate maintained the long plasma half-life of albumin. Theresulting plasma half-life of the conjugate was generally considerablylonger than the plasma half-life of the beneficial therapeutic compoundalone.

Further, albumin has been genetically fused to therapeuticallybeneficial peptides (WO 2001/79271 A and WO2003/59934) with the typicalresult that the fusion has the activity of the therapeuticallybeneficial peptide and a considerably longer plasma half-life than theplasma half-life of the therapeutically beneficial peptides alone.

Otagiri et al (2009), Biol. Pharm. Bull. 32(4), 527-534, discloses morethan 70 albumin variants, of these 25 of these are found to be mutatedin domain III. A natural variant lacking the last 175 amino acids at thecarboxy termini has been shown to have reduced half-life (Andersen et al(2010), Clinical Biochemistry 43, 367-372). Iwao et al (2007) studiedthe half-life of naturally occurring human albumin variants using amouse model, and found that K541E and K560E had reduced half-life, E501Kand E570K had increased half-life and K573E had almost no effect onhalf-life (Iwao, et. al. (2007) B.B.A. Proteins and Proteomics 1774,1582-1590).

Galliano et al (1993) Biochim. Biophys. Acta 1225, 27-32 discloses anatural variant E505K. Minchiotti et al (1990) discloses a naturalvariant K536E. Minchiotti et al (1987) Biochim. Biophys. Acta 916,411-418, discloses a natural variant K574N. Takahashi et al (1987) Proc.Natl. Acad. Sci. USA 84, 4413-4417, discloses a natural variant D550G.Carlson et al (1992). Proc. Nat. Acad. Sci. USA 89, 8225-8229, disclosesa natural variant D550A.

WO2011/051489 and WO2012/150319 disclose a number of point mutations inalbumin which modulate the binding of albumin to FcRn. WO2010/092135discloses a number of point mutations in albumin which increase thenumber of thiols available for conjugation in the albumin, thedisclosure is silent about the affect of the mutations on the binding ofthe albumin to FcRn. WO2011/103076 discloses albumin variants, eachcontaining a substitution in Domain III of HSA. WO2012/112188 disclosesalbumin variants containing substitutions in Domain III of HSA.

Albumin has the ability to bind a number of ligands and these becomeassociated (associates) with albumin. This property has been utilized toextend the plasma half-life of drugs having the ability tonon-covalently bind to albumin. This can also be achieved by binding apharmaceutical beneficial compound, which has little or no albuminbinding properties, to a moiety having albumin binding properties, seereview article and reference therein, Kratz (2008) Journal of ControlledRelease 132, 171-183.

Albumin is used in preparations of pharmaceutically beneficialcompounds, in which such a preparation may be for example, but notlimited to, a nanoparticle or microparticle of albumin. In theseexamples the delivery of a pharmaceutically beneficial compound ormixture of compounds may benefit from alteration in the albumin'saffinity to its receptor where the beneficial compound has been shown toassociate with albumin for the means of delivery. It is not clear whatdetermines the plasma half-life of the formed associates (for examplebut not limited to Levemir®, Kurtzhals P et al. Biochem. J. 1995;312:725-731), conjugates or fusion polypeptides but it appears to be aresult of the combination of the albumin and the selectedpharmaceutically beneficial compound/polypeptide. It would be desirableto be able to control the plasma half-life of given albumin conjugates,associates or albumin fusion polypeptides so that a longer or shorterplasma half-life can be achieved than given by the components of theassociation, conjugation or fusion, in order to be able to design aparticular drug according to the particulars of the indication intendedto be treated.

Albumin is known to accumulate and be catabolised in tumours, it hasalso been shown to accumulate in inflamed joints of rheumatoid arthritissufferers. See review article and reference therein, Kratz (2008)Journal of Controlled Release 132, 171-183. It is envisaged that HSAvariants with increased affinity for FcRn would be advantageous for thedelivery of pharmaceutically beneficial compounds.

It may even be desirable to have variants of albumin that have little orno binding to FcRn in order to provide shorter half-lives or controlledserum pharmacokinetics as described by Kenanova et al (2009) J. Nucl.Med.; 50 (Supplement 2):1582).

Kenanova et al (2010, Protein Engineering, Design & Selection 23(10):789-798; WO2010/118169) discloses a docking model comprising astructural model of domain III of HSA (solved at pH 7 to 8) and astructural model of FcRn (solved at pH 6.4). Kenanova et al disclosesthat positions 464, 505, 510, 531 and 535 in domain III potentiallyinteract with FcRn. The histidines at positions 464, 510 and 535 wereidentified as being of particular interest by Chaudhury et al., (2006,op. cit.) and these were shown to have a significant reduction inaffinity and shorter half-life in mouse by Kenanova (2010, op. cit.).However, the studies of Kenanova et al are limited to domain III of HSAand therefore do not consider HSA in its native intact configuration.Furthermore, the identified positions result in a decrease in affinityfor the FcRn receptor.

The present invention provides further variants having altered bindingaffinity to the FcRn receptor. The albumin moiety or moieties maytherefore be used to tailor the binding affinity to FcRn and/orhalf-life of fusion polypeptides, conjugates, associates, nanoparticlesand compositions comprising the albumin moiety.

SUMMARY OF THE INVENTION

The present invention relates to albumin variants comprising analteration at positions corresponding to positions selected among two ormore of the group consisting of positions 492, 550, 573, 574 and 580 ofthe mature polypeptide of SEQ ID NO: 2 or equivalent positions of otheralbumins or fragments thereof. Position 492 is located in the connectorloop between subdomain DIIIa and subdomain DIIIb. Positions 550, 573,574 and 580 are located in subdomain DIIIb. Subdomain IIIb is locatedproximal to the connector loop between subdomain DIIIa and subdomainDIIIb.

The present invention also relates to isolated polynucleotides encodingthe variants; nucleic acid constructs, vectors, and host cellscomprising the polynucleotides; and methods of producing the variants.

The invention also relates to conjugates or associates comprising thevariant albumin or fragment thereof according to the invention and abeneficial therapeutic moiety or to a fusion polypeptide comprising avariant albumin or fragment thereof of the invention and a fusionpartner polypeptide.

The invention further relates to compositions comprising the variantalbumin, fragment thereof, fusion polypeptide comprising variant albuminor fragment thereof or conjugates comprising the variant albumin orfragment thereof, according to the invention or associates comprisingthe variant albumin or fragment thereof, according to the invention. Thecompositions are preferably pharmaceutical compositions.

The invention further relates to a pharmaceutical composition comprisinga variant albumin, fragment thereof, fusion polypeptide comprisingvariant albumin or fragment thereof or conjugates comprising the variantalbumin or fragment thereof, or associates comprising the variantalbumin or fragment thereof.

The invention also relates to the use of the variants, fragments, fusionpolypeptides, conjugates, associates, nanoparticles and microparticles.

The invention also relates to a method for preparing a variant albumin,fragment thereof, fusion polypeptide comprising variant albumin orfragment thereof or conjugates comprising the variant albumin orfragment thereof, or associates comprising the variant albumin orfragment thereof.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1: Multiple alignment of amino acid sequences of (i) full lengthmature HSA (Hu_(—)1_(—)2_(—)3), (ii) an albumin variant comprisingdomain I and domain III of HSA (Hu_(—)1_(—)3), (iii) an albumin variantcomprising domain II and domain III of HSA (Hu_(—)2_(—)3), (iv)full-length Macaca mulatta albumin (Mac_mul), (v) full-length Rattusnorvegicus albumin (Rat) and (vi) full-length Mus musculus albumin(Mouse). Positions 500, 550 and 573 (relative to full length HSA) areindicated by arrows. In FIG. 1, Domains I, II and III are referred to as1, 2 and 3 (respectively).

FIG. 2: Multiple alignment of amino acid sequences of mature albuminfrom human, sheep, mouse, rabbit and goat and immature albumins fromchimpanzee (“Chimp”), macaque, hamster, guinea pig, rat, cow, horse,donkey, dog, chicken, and pig. The Start and End amino acids of domains1, 2 and 3 (as defined by Dockal et al (The Journal of BiologicalChemistry, 1999, Vol. 274(41): 29303-29310)) are indicated with respectto mature human albumin.

FIG. 3: Conserved groups of amino acids based on their properties.

FIG. 4: Representation of shFcRn-HSA docking model. (A-B) Twoorientations of the complex are shown. Albumin is shown by aspace-filling diagram, FcRn is shown as a ribbon diagram. The corebinding interface of HSA is highlighted in pink (in grey-scale this isseen as the darkest (almost black) region; DI (CBI)), while the areadistally localized from the interface is shown as DII (orange) and DIIIis split into sub-domains DIIIa (in colour, this is cyan) and DIIIb (incolour, this is blue).

DEFINITIONS

Variant: The term “variant” means a polypeptide derived from a parentalbumin by one or more (several) alteration(s), i.e., a substitution,insertion, and/or deletion, at one or more (several) positions. Asubstitution means a replacement of an amino acid occupying a positionwith a different amino acid; a deletion means removal of an amino acidoccupying a position; and an insertion means adding 1 or more, such as1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1-3 amino acids immediatelyadjacent an amino acid occupying a position. In relation tosubstitutions, ‘immediately adjacent’ may be to the N-side (‘upstream’)or C-side (‘downstream’) of the amino acid occupying a position (‘thenamed amino acid’). Therefore, for an amino acid named/numbered ‘X’, theinsertion may be at position ‘X+1’ (‘downstream’) or at position ‘X−1’(‘upstream’).

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Wild-Type Albumin: The term “wild-type” (WT) albumin means albuminhaving the same amino acid sequence as naturally found in an animal orin a human being.

Parent Albumin: The term “parent” or “parent albumin” means an albuminto which an alteration is made by the hand of man to produce the albuminvariants of the invention. The parent may be a naturally occurring(wild-type) polypeptide or an allele thereof, or even a variant thereof.

Albumin: Albumins are proteins and constitute the most abundant proteinin plasma in mammals and albumins from a long number of mammals havebeen characterized by biochemical methods and/or by sequenceinformation. Several albumins, e.g., human serum albumin (HSA), havealso been characterized crystallographically and the structuredetermined (HSA: He X M, Carter D C (July 1992). “Atomic structure andchemistry of human serum albumin”. Nature 358 (6383): 209-15; horsealbumin: Ho, J. X. et al. (2001). X-ray and primary structure of horseserum albumin (Equus caballus) at 0.27-nm resolution. Eur J. Biochem.215(1):205-12).

The term “albumin” means a protein having the same and/or very similarthree dimensional (tertiary) structure as HSA or HSA domains and hassimilar properties to HSA or to the relevant domains. Similar threedimensional structures are for example the structures of the albuminsfrom the species mentioned herein. Some of the major properties ofalbumin are i) its ability to regulate plasma volume (oncotic activity),ii) a long plasma half-life of around 19 days±5 days, iii) binding toFcRn, iv) ligand-binding, e.g. binding of endogenous molecules such asacidic, lipophilic compounds including bilirubin, fatty acids, hemin andthyroxine (see also table 1 of Kragh-Hansen et al, 2002, Biol. Pharm.Bull. 25, 695, hereby incorporated by reference), v) binding of smallorganic compounds with acidic or electronegative features e.g. drugssuch as warfarin, diazepam, ibuprofen and paclitaxel (see also table 1of Kragh-Hansen et al, 2002, Biol. Pharm. Bull. 25, 695, herebyincorporated by reference). Not all of these properties need to befulfilled in order to characterize a protein or fragment as an albumin.If a fragment, for example, does not comprise a domain responsible forbinding of certain ligands or organic compounds the variant of such afragment will not be expected to have these properties either.

Albumins have generally a long plasma half-life of approximately 20 daysor longer, e.g., HSA has a plasma half-life of 19 days. It is known thatthe long plasma half-life of HSA is mediated via interaction with itsreceptor FcRn, however, an understanding or knowledge of the exactmechanism behind the long half-life of HSA is not essential for theinvention.

As examples of albumin proteins, more specifically albumin proteinswhich may be used as parent ‘backbones’ as a starting point for makingthe albumin variants according to the invention, can be mentioned humanserum albumin (e.g. AAA98797 or P02768-1, SEQ ID NO: 2 (mature), SEQ IDNO: 4 (immature)), primate serum albumin, (such as chimpanzee serumalbumin (e.g. predicted sequence XP_(—)517233.2 SEQ ID NO: 5), gorillaserum albumin or macaque serum albumin (e.g. NP_(—)001182578, SEQ ID NO:6), rodent serum albumin (such as hamster serum albumin (e.g. A6YF56,SEQ ID NO: 7), guinea pig serum albumin (e.g. Q6WDN9-1, SEQ ID NO: 8),mouse serum albumin (e.g. AAH49971 or P07724-1 Version 3, SEQ ID NO: 9)and rat serum albumin (e.g. AAH85359 or P02770-1 Version 2, SEQ ID NO:10))), bovine serum albumin (e.g. cow serum albumin P02769-1, SEQ ID NO:11), equine serum albumin such as horse serum albumin (e.g. P35747-1,SEQ ID NO: 12) or donkey serum albumin (e.g. Q5XLE4-1, SEQ ID NO: 13),rabbit serum albumin (e.g. P49065-1 Version 2, SEQ ID NO: 14), goatserum albumin (e.g. ACF10391, SEQ ID NO: 15), sheep serum albumin (e.g.P14639-1, SEQ ID NO: 16), dog serum albumin (e.g. P49822-1, SEQ ID NO:17), chicken serum albumin (e.g. P19121-1 Version 2, SEQ ID NO: 18) andpig serum albumin (e.g. P08835-1 Version 2, SEQ ID NO: 19) or apolypeptide having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96,97, 98 or at least 99% amino acid identity to such an albumin. Theparent or reference albumin may be an artificial variant such as HSA.K573P (SEQ ID NO: 3) or a chimeric albumin such as the N-terminal of HSAand the C-terminal of macaca albumin (SEQ ID NO: 20), N-terminal of HSAand the C-terminal of mouse albumin (SEQ ID NO: 21), N-terminal of HSAand the C-terminal of rabbit albumin (SEQ ID NO: 22), N-terminal of HSAand the C-terminal of sheep albumin (SEQ ID NO: 23).

Other examples of albumin, which are also included in the scope of thisapplication, include ovalbumin (e.g. P01012.pro: chicken ovalbumin;O73860.pro: turkey ovalbumin).

HSA as disclosed in SEQ ID NO: 2 or any naturally occurring allelethereof, is the preferred parent albumin according to the invention. HSAis a protein consisting of 585 amino acid residues and has a molecularweight of 67 kDa. In its natural form it is not glycosylated. Theskilled person will appreciate that natural alleles may exist havingessentially the same properties as HSA but having one or more (several)amino acid changes compared to SEQ ID NO: 2, and the inventors alsocontemplate the use of such natural alleles as parent albumin accordingto the invention.

The parent albumin, a fragment thereof, or albumin part of a fusionpolypeptide comprising albumin or a fragment thereof according to theinvention preferably has a sequence identity to the sequence of HSAshown in SEQ ID NO: 2 of at least 60%, preferably at least 70%,preferably at least 80%, preferably at least 85%, preferably at least86%, preferably at least 87%, preferably at least 88%, preferably atleast 89%, preferably at least 90%, preferably at least 91%, preferablyat least 92%, preferably at least 93%, preferably at least 94%,preferably at least 95%, more preferred at least 96%, more preferred atleast 97%, more preferred at least 98% and most preferred at least 99%.It is preferred that the parent albumin maintains at least one of themajor properties of albumin or a similar tertiary structure as analbumin, such as HSA. The sequence identity may be over the full-lengthof SEQ ID NO: 2 or over a molecule consisting or comprising of afragment such as one or more (several) domains of SEQ ID NO: 2 such as amolecule consisting of or comprising domain III (e.g. SEQ ID NO: 27), amolecule consisting of or comprising domain II and domain III (e.g. SEQID NO: 25), a molecule consisting of or comprising domain I and domainIII (e.g. SEQ ID NO: 24), a molecule consisting of or comprising twocopies of domain III (e.g. SEQ ID NO: 26), a molecule consisting of orcomprising three copies of domain III (e.g. SEQ ID NO: 28) or a moleculeconsisting of or comprising domain I and two copies of domain III (e.g.SEQ ID NO: 29).

The parent preferably comprises or consists of the amino acid sequenceof SEQ ID NO: 4 (immature sequence of HSA) or SEQ ID NO: 2 (maturesequence of HSA).

In another embodiment, the parent is an allelic variant of the maturepolypeptide of SEQ ID NO: 2.

The parent albumin may be encoded by a polynucleotide that hybridizesunder very low stringency conditions, low stringency conditions, mediumstringency conditions, medium-high stringency conditions, highstringency conditions, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1 or (ii) thefull-length complementary strand of (i) (J. Sambrook, E. F. Fritsch, andT. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition,Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe amino acid sequence of SEQ ID NO: 2 or a fragment thereof, may beused to design nucleic acid probes to identify and clone DNA encoding aparent from strains of different genera or species according to methodswell known in the art. In particular, such probes can be used forhybridization with the genomic or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least14, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labelled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the invention.

A genomic DNA or cDNA library prepared from such other organisms may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that is homologouswith SEQ ID NO: 1 or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the invention, hybridization indicates that thepolynucleotide hybridizes to a labelled nucleotide probe correspondingto the polynucleotide shown in SEQ ID NO: 1, its complementary strand,or a subsequence thereof, under low to very high stringency conditions.Molecules to which the probe hybridizes can be detected using, forexample, X-ray film or any other detection means known in the art.

The nucleic acid probe may comprise or consist of the mature polypeptidecoding sequence of SEQ ID NO: 1, i.e. nucleotides 1 to 1785 of SEQ IDNO: 1. The nucleic acid probe may comprise or consist of apolynucleotide that encodes the polypeptide of SEQ ID NO: 2 or afragment thereof.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as pre-hybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and either 25% formamide for very lowand low stringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed three times each for15 minutes using 2×SSC, 0.2% SDS at 45° C. (very low stringency), 50° C.(low stringency), 55° C. (medium stringency), 60° C. (medium-highstringency), 65° C. (high stringency), or 70° C. (very high stringency).

For short probes that are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as pre-hybridization andhybridization at about 5° C. to about 10° C. below the calculated T_(n),using the calculation according to Bolton and McCarthy (1962, Proc.Natl. Acad. Sci. USA 48: 1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA perml following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed once in 6×SCC plus0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5°C. to 10° C. below the calculated T_(m).

The parent may be encoded by a polynucleotide with a sequence identityto the mature polypeptide coding sequence of SEQ ID NO: 1 of at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, which encodes a polypeptide which isable to function as an albumin. In an embodiment, the parent is encodedby a polynucleotide comprising or consisting of SEQ ID NO: 1.

Albumin moiety: The albumin part of a fusion polypeptide, conjugate,associate, nanoparticle or composition comprising the albumin variant orfragment thereof according to the invention, may be referred to as an‘albumin moiety’ or ‘albumin component’. A polypeptide according to theinvention may comprise or consist of an albumin moiety.

FcRn and shFcRn: The term “FcRn” means the human neonatal Fc receptor(FcRn). shFcRn is a soluble recombinant form of FcRn. hFcRn is aheterodimer of SEQ ID NO: 30 (truncated heavy chain of the majorhistocompatibility complex class I-like Fc receptor (FCGRT)) and SEQ IDNO: 31 (beta-2-microglobulin). Together, SEQ ID NO: 30 and 31 formhFcRn.

Isolated variant: The term “isolated variant” means a variant in a formor environment which does not occur in nature. Non-limiting examples ofisolated variants include (1) any non-naturally occurring variant, (2)any variant that is at least partially removed from one or more(several) or all of the naturally occurring constituents with which itis associated in nature; (3) any variant modified by the hand of manrelative to the polypeptide from which it is derived (e.g. thepolypeptide from which it is derived as found in nature); or (4) anyvariant modified by increasing the amount of the variant e relative toother components with which it is naturally associated (e.g., multiplecopies of a gene encoding the substance; use of a stronger promoter thanthe promoter naturally associated with the gene encoding the substance).An isolated variant may be present in a fermentation broth sample.

Substantially pure variant: The term “substantially pure variant” meansa preparation that contains at most 10%, at most 8%, at most 6%, at most5%, at most 4%, at most 3%, at most 2%, at most 1%, and at most 0.5% byweight of other polypeptide material with which it is natively orrecombinantly associated. Preferably, the variant is at least 92% pure,e.g., at least 94% pure, at least 95% pure, at least 96% pure, at least97% pure, at least 98% pure, at least 99%, at least 99.5% pure, and 100%pure by weight of the total polypeptide material present in thepreparation. Purity may be determined by SDS-PAGE or GP-HPLC. Thevariants of the invention are preferably in a substantially pure form.This can be accomplished, for example, by preparing the variant bywell-known recombinant methods and by purification methods.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. The mature polypeptide may be aminoacids 1 to 585 of SEQ ID NO: 2, e.g. with the inclusion of alterationsaccording to the invention and/or any post-translational modifications.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature albuminpolypeptide. The mature polypeptide coding sequence may be nucleotides 1to 1758 of SEQ ID NO: 1e.g. with the alterations required to encode avariant according to the invention.

Sequence Identity:

The relatedness between two amino acid sequences or between twonucleotide sequences is described by the parameter “sequence identity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the—nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the—nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Fragment: The term “fragment” means a polypeptide having one or more(several) amino acids deleted from the amino and/or carboxyl terminus ofan albumin and/or an internal region of albumin that has retained theability to bind to FcRn. Fragments may consist of one uninterruptedsequence derived from HSA or it may comprise two or more sequencesderived from HSA. The fragments according to the invention have a sizeof more than approximately 20 amino acid residues, preferably more than30 amino acid residues, more preferred more than 40 amino acid residues,more preferred more than 50 amino acid residues, more preferred morethan 75 amino acid residues, more preferred more than 100 amino acidresidues, more preferred more than 200 amino acid residues, morepreferred more than 300 amino acid residues, even more preferred morethan 400 amino acid residues and most preferred more than 500 amino acidresidues. A fragment may comprise or consist of one more domains ofalbumin such as DI+DII, DI+DIII, DII+DIII, DIII+DIII, DI+DIII+DIII,DIII+DIII+DIII, or fragments of such domains or combinations of domains.

Domains I, II and III may be defined with reference to HSA (SEQ ID NO:2). For example, HSA domain I may consist of or comprise amino acids 1to 194 (±1 to 15 amino acids) of SEQ ID NO: 2, HSA domain II may consistof or comprise amino acids 192 (±1 to 15 amino acids) to 387 (±1 to 15amino acids) of SEQ ID NO: 2 and domain III may consist of or compriseamino acid residues 381 (±1 to 15 amino acids) to 585 (±1 to 15 aminoacids) of SEQ ID NO: 2. “±1 to 15 amino acids” means that the residuenumber may deviate by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or15 amino acids to the C-terminus and/or to the N-terminus of the statedamino acid position. Examples of domains I, II and III are described byDockal et al (The Journal of Biological Chemistry, 1999, Vol. 274(41):29303-29310) and Kjeldsen et al (Protein Expression and Purification,1998, Vol 13: 163-169) and are tabulated below.

Amino acid residues of HSA domains I, II and III with reference to SEQID NO: 2 Dockal et al Kjeldsen et al Domain I  1 to 197  1 to 192 DomainII 189 to 385 193 to 382 Domain III 381 to 585 383 to 585

The skilled person can identify domains I, II and III in non-humanalbumins by amino acid sequence alignment with HSA, for example usingthe Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.Biol. 48: 443-453) as implemented in the Needle program of the EMBOSSpackage (EMBOSS: The European Molecular Biology Open Software Suite,Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0or later. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. Other suitable software includes MUSCLE ((Multiplesequence comparison by log-expectation, Robert C. Edgar, Version 3.6,http://www.drive5.com/muscle; Edgar (2004) Nucleic Acids Research 32(5),1792-97 and Edgar (2004) BMC Bioinformatics, 5(1):113) which may be usedwith the default settings as described in the User Guide (Version 3.6,September 2005). Versions of MUSCLE later than 3.6 may also be used forany aspect of the invention). Examples of suitable alignments areprovided in FIGS. 1 and 2.

It is preferred that domains have at least 70, 75, 80, 85, 90, 95, 96,97, 98, 99, 99.5% identity or 100% identity to Domain I, II or III ofHSA (SEQ ID NO: 2).

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of its translatedpolypeptide product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG, and TGA. The coding sequence may bea DNA, cDNA, synthetic, or recombinant polynucleotide.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic cell. cDNA lacks intron sequences that may be presentin the corresponding genomic DNA. The initial, primary RNA transcript isa precursor to mRNA that is processed through a series of steps,including splicing, before appearing as mature spliced mRNA.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic. The term nucleic acid construct issynonymous with the term “expression cassette” when the nucleic acidconstruct contains the control sequences required for expression of acoding sequence of the invention.

Control sequences: The term “control sequences” means all nucleic acidsequences necessary for the expression of a polynucleotide encoding avariant of the invention. Each control sequence may be native (i.e. fromthe same gene) or foreign (i.e. from a different gene) to thepolynucleotide encoding the variant or native or foreign to each other.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs the expression of the coding sequence.

Expression: The term “expression” includes any step involved in theproduction of the variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Plasma half-life: Plasma half-life is ideally determined in vivo insuitable individuals. However, since it is time consuming and expensiveand there inevitable are ethical concerns connected with doingexperiments in animals or man it is desirable to use an in vitro assayfor determining whether plasma half-life is extended or reduced. It isknown that the binding of albumin to its receptor FcRn is important forplasma half-life and the correlation between receptor binding and plasmahalf-life is that a higher affinity of albumin to its receptor leads tolonger plasma half-life. Thus for the invention a higher affinity ofalbumin to FcRn is considered indicative of an increased plasmahalf-life and a lower affinity of albumin to its receptor is consideredindicative of a reduced plasma half-life.

In this application and claims the binding of albumin to its receptorFcRn is described using the term affinity and the expressions “stronger”or “weaker”. Thus, it should be understood that a molecule having ahigher affinity to FcRn than HSA is considered to bind stronger to FcRnthan HSA and a molecule having a lower affinity to FcRn than HSA isconsidered to bind weaker to FcRn than HSA.

The terms “longer plasma half-life” or “shorter plasma half-life” andsimilar expressions are understood to be in relationship to thecorresponding parent or reference or corresponding albumin molecule.Thus, a longer plasma half-life with respect to a variant albumin of theinvention means that the variant has longer plasma half-life than thecorresponding albumin having the same sequences except for thealteration(s) described herein, e.g. at two or more positionscorresponding to 492, 550, 573, 574 and 580 of HSA (SEQ ID NO: 2).

Reference: a reference is an albumin, fusion, conjugate, composition,associate or nanoparticle to which an albumin variant, fusion,conjugate, composition, associate or nanoparticle is compared. Thereference may comprise or consist of full length albumin (such as HSA ora natural allele thereof) or a fragment thereof. A reference may also bereferred to as a ‘corresponding’ albumin, fusion, conjugate,composition, associate or nanoparticle to which an albumin variant,fusion, conjugate, composition, associate or nanoparticle is compared. Areference may comprise or consist of HSA (SEQ ID NO: 2) or a fragment,fusion, conjugate, associate, nanoparticle or microparticle thereof.Preferably, the reference is identical to the polypeptide, fusionpolypeptide, conjugate, composition, associate, nanoparticle ormicroparticle according to the invention (“being studied”) with theexception of the albumin moiety. Preferably the albumin moiety of thereference comprises or consists of an albumin (e.g. HSA, SEQ ID NO: 2)or a fragment thereof. The amino acid sequence of the albumin moiety ofthe reference may be longer than, shorter than or, preferably, the same(±1 to 15 amino acids) length as the amino sequence of the albuminmoiety of the polypeptide, fusion polypeptide, conjugate, composition,associate, nanoparticle or microparticle according to the invention(“being studied”).

Equivalent amino acid positions: Throughout this specification aminoacid positions are defined in relation to full-length mature human serumalbumin (i.e. without leader sequence, SEQ ID NO: 2). However, theskilled person understands that the invention also relates to variantsof non-human albumins (e.g. those disclosed herein) and/or fragments ofa human or non-human albumin. Equivalent positions can be identified infragments of human serum albumin, in animal albumins and in fragments,fusions and other derivative or variants thereof by comparing amino acidsequences using pairwise (e.g. ClustalW) or multiple (e.g. MUSCLE)alignments. For example, FIG. 1 shows that positions equivalent to 500,550 and 573 in full length human serum albumin are easily identified infragments of human serum albumin and in albumins of other species.Positions 500, 550 and 573 are indicated by arrows. Further details areprovided in Table 1 below.

TABLE 1 Example of identification of equivalent positions in HSA, animalalbumins and albumin fragments Albumin Position equivalent to humanserum albumin Organism (accession Full length or Fragment Total lengthof (native amino acid): number of protein) fragment details matureprotein 500 (K) 550 (D) 573 (K) Homo sapiens Full length — 585 500 (K)550 (D) 573 (K) (AAA98797) Homo sapiens Fragment DI, DIII 399 314 (K)364 (D) 387 (K) Homo sapiens Fragment DI, DIII 403 318 (K) 368 (D) 391(K) Macaca mulatta Full length — 584 500 (K) 550 (N) 573 (P) (NP_001182578) Rattus norvegicus Full length — 584 500 (K) 550 (D) 573(P)  (AAH85359) Mus musculus Full length — 584 500 (K) 550 (D) 573 (P) (AAH49971)

FIG. 1 was generated by MUSCLE using the default parameters includingoutput in ClustalW 1.81 format. The raw output data was shaded usingBoxShade 3.21 (http://www.ch.embnet.orq/software/BOX form.html) usingOutput Format: RTF_new; Font Size: 10; Consensus Line: no consensusline; Fraction of sequences (that must agree for shading): 0.5; Inputsequence format: ALN. Therefore, throughout this specification aminoacid positions defined in human serum albumin also apply to equivalentpositions in fragments, derivatives or variants and fusions of humanserum albumin, animals from other species and fragments and fusionsthereof. Such equivalent positions may have (i) a different residuenumber in its native protein and/or (ii) a different native amino acidin its native protein.

Likewise, FIG. 2 shows that equivalent positions can be identified infragments (e.g. domains) of an albumin with reference to SEQ ID NO: 2(HSA).

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide disclosedin SEQ ID NO: 2 is used to determine the corresponding amino acidresidue in another albumin. The amino acid sequence of another albuminis aligned with the mature polypeptide disclosed in SEQ ID NO: 2, andbased on the alignment, the amino acid position number corresponding toany amino acid residue in the mature polypeptide disclosed in SEQ ID NO:2 is determined using the Needleman-Wunsch algorithm (Needleman andWunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleprogram of the EMBOSS package (EMBOSS: The European Molecular BiologyOpen Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),preferably version 5.0.0 or later. The parameters used are gap openpenalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSSversion of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in anotheralbumin can be determined or confirmed by an alignment of multiplepolypeptide sequences using several computer programs including, but notlimited to, MUSCLE (multiple sequence comparison by log-expectation;version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32:1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, NucleicAcids Research 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research33: 511-518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh etal., 2009, Methods in Molecular Biology 537:_(—)39-64; Katoh and Toh,2010, Bioinformatics 26:_(—)1899-1900), and EMBOSS EMMA employingClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research22: 4673-4680), using their respective default parameters.

When the other polypeptide (or protein) has diverged from the maturepolypeptide of SEQ ID NO: 2 such that traditional sequence-basedcomparison fails to detect their relationship (Lindahl and Elofsson,2000, J. Mol. Biol. 295: 613-615), other pairwise sequence comparisonalgorithms can be used. Greater sensitivity in sequence-based searchingcan be attained using search programs that utilize probabilisticrepresentations of polypeptide families (profiles) to search databases.For example, the PSI-BLAST program generates profiles through aniterative database search process and is capable of detecting remotehomologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Evengreater sensitivity can be achieved if the family or superfamily for thepolypeptide has one or more representatives in the protein structuredatabases. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881) utilizeinformation from a variety of sources (PSI-BLAST, secondary structureprediction, structural alignment profiles, and solvation potentials) asinput to a neural network that predicts the structural fold for a querysequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol.313: 903-919, can be used to align a sequence of unknown structure withthe superfamily models present in the SCOP database. These alignmentscan in turn be used to generate homology models for the polypeptide, andsuch models can be assessed for accuracy using a variety of toolsdeveloped for that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the albumin variants of the present invention, thenomenclature described below is adapted for ease of reference. Theaccepted IUPAC single letter or three letter amino acid abbreviation isemployed. The term ‘point mutation’ and/or ‘alteration’ includesdeletions, insertions and substitutions.

Substitutions.

For an amino acid substitution, the following nomenclature is used:Original amino acid, position, substituted amino acid. Accordingly, thesubstitution of threonine at position 226 with alanine is designated as“Thr226Ala” or “T226A”. Multiple mutations (or alterations) areseparated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+S411F”, representing substitutions at positions 205 and 411 ofglycine (G) with arginine (R) and serine (S) with phenylalanine (F),respectively. The Figures also use (“/”), e.g., “E492T/N503D” thisshould be viewed as interchangeable with (“+”).

Deletions.

For an amino acid deletion, the following nomenclature is used: Originalamino acid, position*. Accordingly, the deletion of glycine at position195 is designated as “Glyl95*” or “G195*”. Multiple deletions areseparated by addition marks (“+”), e.g., “Glyl95*+Ser411*” or“G195*+S411*”.

Insertions.

As disclosed above, an insertion may be to the N-side (‘upstream’,‘X−1’) or C-side (‘downstream’, ‘X+1’) of the amino acid occupying aposition (‘the named (or original) amino acid’, ‘X’).

For an amino acid insertion to the C-side (‘downstream’, ‘X+1’) of theoriginal amino acid (‘X’), the following nomenclature is used: Originalamino acid, position, original amino acid, inserted amino acid.Accordingly the insertion of lysine after glycine at position 195 isdesignated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G-K-A

For an amino acid insertion to the N-side (‘upstream’, ‘X−1’) of theoriginal amino acid (X), the following nomenclature is used: Originalamino acid, position, inserted amino acid, original amino acid.Accordingly the insertion of lysine (K) before glycine (G) at position195 is designated “Gly195LysGly” or “G195KG”. An insertion of multipleamino acids is designated [Original amino acid, position, inserted aminoacid #1, inserted amino acid #2; etc., original amino acid]. Forexample, the insertion of lysine (K) and alanine (A) before glycine atposition 195 is indicated as “Gly195LysAlaGly” or “G195KAG”. In suchcases the inserted amino acid residue(s) are numbered by the addition oflower case letters with prime to the position number of the amino acidresidue following the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195a′ 195b′ 195 G K-A-G

Multiple Alterations.

Variants comprising multiple alterations are separated by addition marks(“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing asubstitution of arginine and glycine at positions 170 and 195 tyrosineand glutamic acid, respectively.

Different Alterations.

Where different alterations can be introduced at a position, thedifferent alterations are separated by a comma, e.g., “Arg170Tyr,Glu”represents a substitution of arginine at position 170 with tyrosine orglutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” designates thefollowing variants: “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”,“Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to albumin variants, comprising analteration at two or more positions selected among the group consistingof positions 492, 550, 573, 574 and 580 of the mature polypeptide of SEQID NO: 2, or at equivalent positions in other albumins or fragmentsthereof.

Variants

A first aspect of the invention provides polypeptides which are variantalbumins or fragments thereof, or fusion polypeptides comprising thevariant albumin or fragments thereof, of a parent albumin, comprisingalterations at two or more positions corresponding to positions selectedamong the group consisting of positions 492, 550, 573, 574 and 580 ofthe mature polypeptide of SEQ ID NO: 2. It is preferred that the two ormore alterations comprise alterations at positions corresponding to thefollowing positions in SEQ ID NO: 2:

(a) 492 and 580; and/or(b) 492 and 574; and/or(c) 492 and 550; and/or(d) 550 and 573; and/or(e) 550 and 574; and/or(f) 550 and 580; and/orand/or that(g) the two or more alterations comprise:

-   -   an alteration to generate at a position corresponding to        position 492 of SEQ ID NO: 2 an amino acid from the group        consisting of A, C, D, F, H, I, K, L, M, N, P, Q, R, S, T, V, W,        Y, preferably D and an alteration to generate at a position        corresponding to position 573 of SEQ ID NO: 2 to an amino acid        from the group consisting of C, D, E, F, G, H, I, L, M, N, Q, R,        S, T, V, W, Y, preferably Y, W or H or    -   an alteration to generate at a position corresponding to        position 492 of SEQ ID NO: 2 a G and an alteration to generate        at a position corresponding to position 573 an A or P and an        additional alteration at a position selected from the group        consisting of 550,574 and 580; and/or        (h) the two or more alterations comprise:    -   an alteration to generate at a position corresponding to        position 573 of SEQ ID NO: 2 an amino acid from the group        consisting of A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W,        Y, preferably Y, W or H and an alteration to generate at a        position corresponding to position 574 of SEQ ID NO: 2 an amino        acid from the group consisting of A, C, D, E, F, G, H, I, L, M,        P, Q, R, S, T, V, W, Y, H, D, F, G, N, S or Y, more preferably        H, D, F or G, most preferably H or    -   an alteration to generate at a position corresponding to        position 573 of SEQ ID NO: 2 a P and an alteration to generate        at a position corresponding to position 574 of SEQ ID NO: 2 an N        and an additional alteration at a position selected from the        group consisting of 492, 550, and 580; and/or        (i) the two or more alterations comprise:    -   an alteration to generate at a position corresponding to        position 573 of SEQ ID NO: 2 an amino acid from the group        consisting of A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W,        Y, preferably Y, W or H and an alteration to generate at a        position corresponding to position 580 of SEQ ID NO: 2 an amino        acid from the group consisting of C, D, E, F, G, H, I, L, M, N,        P, R, S, T, V, W, Y or    -   an alteration to generate at a position corresponding to        position 573 of SEQ ID NO: 2 a P and an alteration to generate        at a position corresponding to position 580 of SEQ ID NO: 2 a K        and an additional alteration at a position selected from the        group consisting of 492, 550, and 574; and/or        (j) the two or more alterations comprise:    -   an alteration to generate at a position corresponding to        position 574 of SEQ ID NO: 2 an amino acid from the group        consisting of A, C, D, E, F, G, H, I, L, M, P, Q, R, S, T, V, W,        Y, H, D, F, G, N, S or Y, more preferably H and an alteration to        generate at a position corresponding to position 580 of SEQ ID        NO: 2 to an amino acid from the group consisting of A, C, D, E,        F, G, H, I, L, M, N, P, R, S, T, V, W, Y or    -   an alteration to generate at a position corresponding to        position 574 of SEQ ID NO: 2 an N and an alteration to generate        at a position corresponding to position 580 of SEQ ID NO: 2 a K        and an additional alteration at a position selected from the        group consisting of 492, 550, and 573

The polypeptide may comprise, three or more, four or more or five ormore alterations as described in paragraphs (a), (b), (c), (d), (e),(f), (g), (h), (i) and (j).

A preferred alteration is a substitution.

It is preferred that the parent albumin and/or the variant albumincomprises or consists of:

(a) a polypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 2;

(b) a polypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, or (ii) the full-length complement of (i);

(c) a polypeptide encoded by a polynucleotide having at least 60%identity to the mature mature polypeptide coding sequence of SEQ ID NO:1; and/or

(d) a fragment of the mature polypeptide of SEQ ID NO: 2.

The variants of albumin or fragments thereof or fusion polypeptidescomprising albumin or fragments thereof comprise alterations, such assubstitutions, deletions or insertions at two or more of positionsselected among the group consisting of positions 492, 550, 573, 574 and580 of the mature polypeptide of SEQ ID NO: 2 or in equivalent positionsof other albumins or variants or fragments thereof. A stop codon may beintroduced in addition to the alterations described herein and ifintroduced is at position 574 or further downstream (e.g. in SEQ ID NO:2 it is introduced at from position 574 to 585).

The variant albumin, a fragment thereof, or albumin part of a fusionpolypeptide comprising variant albumin or a fragment thereof accordingto the invention has generally a sequence identity to the sequence ofHSA shown in SEQ ID NO: 2 of at least 60%, preferably at least 70%,preferably at least 80%, preferably at least 85%, preferably at least90%, more preferred at least 95%, more preferred at least 96%, morepreferred at least 97%, more preferred at least 98% and most preferredat least 99%. The variant has less than 100% identity to SEQ ID NO: 2.

The variant albumin, a fragment thereof, or albumin part of a fusionpolypeptide comprising variant albumin or a fragment thereof accordingto the invention has generally a sequence identity to the sequence ofthe parent albumin of at least 60%, preferably at least 70%, preferablyat least 80%, preferably at least 85%, preferably at least 90%, morepreferred at least 95%, more preferred at least 96%, more preferred atleast 97%, more preferred at least 98% and most preferred at least 99%.The variant has less than 100% identity to the sequence of the parentalbumin.

In one aspect, the number of alterations in the variants of theinvention is 1 to 20, e.g., 1 to 10 and 1 to 5, such as 1, 2, 3, 4, 5,6, 7, 8, 9 or 10 alterations relative to SEQ ID NO: 2 or relative to thesequence of the parent albumin.

At position 492 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof), it is preferred that thealteration is a substitution, such as from the native amino acid to A,C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, more preferredto G, D, F, H, M or R, even more preferred to G, or D and most preferredto G. In SEQ ID NO: 2 the native amino acid at position 492 is E,therefore a substitution to E is not preferred.

At position 550 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof), it is preferred that thealteration is a substitution, such as from the native amino acid to A,C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, more preferredto K, L, M, E or R, even more preferred to K, L or M and most preferredto K. In SEQ ID NO: 2 the native amino acid at position 550 is D,therefore a substitution to D is not preferred.

At position 573 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof), it is preferred that thealteration is a substitution, such as from the native amino acid to A,C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, more preferredto P, Y, W, H, F, T, I or V, even more preferred to P, Y or W and mostpreferred to P. In SEQ ID NO: 2 the native amino acid at position 573 isK, therefore a substitution to K is not preferred.

At position 574 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof), it is preferred that thealteration is a substitution, such as from the native amino acid to A,C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, more preferredto H, G, D, F, N, S or Y, even more preferred to D, F, G or H and mostpreferred to H. In SEQ ID NO: 2 the native amino acid at position 574 isK, therefore a substitution to K is not preferred.

At position 580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof), it is preferred that thealteration is a substitution, such as from the native amino acid to A,C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, more preferredto K or R, most preferred to K. In SEQ ID NO: 2 the native amino acid atposition 580 is Q, therefore a substitution to Q is not preferred.

A variant albumin may comprise alterations at positions corresponding topositions 492+550 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 492D+550K (e.g. SEQ ID NO: 231), or 492G+550K (e.g. SEQ ID NO:240).

A variant albumin may comprise alterations at positions corresponding topositions 492+573 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 492F+573P (e.g. SEQ ID NO: 109), 492G+573P (e.g. SEQ ID NO:110), 492H+573P (e.g. SEQ ID NO: 111) or 492R+573P (e.g. SEQ ID NO: 113)or more preferably 492D+573P (e.g. SEQ ID NO: 108). However, it ispreferred that the variant does not consist of SEQ ID NO: 2 with onlyalterations 492G+573A, 492G+573A, 492G+N503K+573A, 492G+N503H-F573A,492G+573P, 492G+N503K+573P or 492G+N503H+573P.

A variant albumin may comprise alterations at positions corresponding topositions 492+574 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 492D+574H (e.g. SEQ ID NO: 232), 492G+574H

(e.g. SEQ ID NO: 241) or 492D+574H (e.g. SEQ ID NO: 232).

A variant albumin may comprise alterations at positions corresponding topositions 492+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof).

A variant albumin may comprise alterations at positions corresponding topositions 550+573 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 550K+573P (e.g. SEQ ID NO: 117).

A variant albumin may comprise alterations at positions corresponding topositions 550+574 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 550K+574H (e.g. SEQ ID NO: 130), 550M+574H (e.g. SEQ ID NO:249), 550M+574H (e.g. SEQ ID NO: 249) or 550L+574H (e.g. SEQ ID NO:245).

A variant albumin may comprise alterations at positions corresponding topositions 550+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 550K+580K (e.g. SEQ ID NO: 131), 550M+580K (e.g. SEQ ID NO:251) or 550L+580K (e.g. SEQ ID NO: 247).

A variant albumin may comprise alterations at positions corresponding topositions 573+574 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 574D+573P (e.g. SEQ ID NO: 121), 574F+573P (e.g. SEQ ID NO:122), 574G+573P (e.g. SEQ ID NO: 123), 574H+573P (e.g. SEQ ID NO: 124),574N+573P (e.g. SEQ ID NO: 125) or 5745+573P (e.g. SEQ ID NO: 126). Itis preferred that the variant does not consist of SEQ ID NO: 2 with onlyalterations K573P+K574N+A577T+A578R+S579C+Q580K+A581 D+G584A.

A variant albumin may comprise alterations at positions corresponding topositions 573+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 580K+573P (e.g. SEQ ID NO: 128) or 580R+573P (e.g. SEQ ID NO:129). However, it is preferred that the variant does not consist of SEQID NO: 2 with only alterations K573P+A577E+A578S+Q580K+A582T.

A variant albumin may comprise alterations at positions corresponding topositions 574+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof).

A variant albumin may comprise alterations at positions corresponding topositions 492+550+573 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 492G+550K+573P (e.g. SEQ ID NO: 254) or 492D+550K+573P (e.g.SEQ ID NO: 253).

A variant albumin may comprise alterations at positions corresponding topositions 492+550+574 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 492G+550K+574H (e.g. SEQ ID NO: 255).

A variant albumin may comprise alterations at positions corresponding topositions 492+550+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 492D+550K+580K (e.g. SEQ ID NO: 258) or 492G+550K+580K (e.g.SEQ ID NO: 259).

A variant albumin may comprise alterations at positions corresponding topositions 492+573+574 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 492D+573P+574H (e.g. SEQ ID NO: 233).

A variant albumin may comprise alterations at positions corresponding topositions 492+573+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 492D+573P+580K (e.g. SEQ ID NO: 234) or 492G+573P+580K (e.g.SEQ ID NO: 242).

A variant albumin may comprise alterations at positions corresponding topositions 492+574+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 492D+574H+580K (SEQ ID NO: 262) or 492G+574H+580K (e.g. SEQ IDNO: 263).

A variant albumin may comprise alterations at positions corresponding topositions 50+573+574 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 550K+574H+573P (e.g. SEQ ID NO: 131), 550L+573P+574H (e.g. SEQID NO: 246) or 550M+573P+574H (e.g. SEQ ID NO: 250).

A variant albumin may comprise alterations at positions corresponding topositions 550+573+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 550L+573P+580K (e.g. SEQ ID NO: 248) or 550M+573P+580K (e.g.SEQ ID NO: 252).

A variant albumin may comprise alterations at positions corresponding topositions 550+574+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof).

A variant albumin may comprise alterations at positions corresponding topositions 573+574+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 574H+580K+573P (e.g. SEQ ID NO: 135).

A variant albumin may comprise alterations at positions corresponding topositions 492+550+573+574 of SEQ ID NO: 2 (or equivalent position ofother albumins or variants or fragments thereof). Such alterations maycomprise 492G+550K+573P+574H (e.g. SEQ ID NO: 257) or492D+550K+573P+574H (e.g. SEQ ID NO: 256).

A variant albumin may comprise alterations at positions corresponding topositions 492+550+573+580 of SEQ ID NO: 2 (or equivalent position ofother albumins or variants or fragments thereof). Such alterations maycomprise 492D+550K+573P+580K (e.g. SEQ ID NO: 260) or492G+550K+573P+580K (e.g. SEQ ID NO: 261).

A variant albumin may comprise alterations at positions corresponding topositions 492+550+574+580 of SEQ ID NO: 2 (or equivalent position ofother albumins or variants or fragments thereof).

A variant albumin may comprise alterations at positions corresponding topositions 492+573+574+580 of SEQ ID NO: 2 (or equivalent position ofother albumins or variants or fragments thereof). Such alterations maycomprise 492D+573P+574H+580K (e.g. SEQ ID NO: 114) or492G+573P+574H+580K (e.g. SEQ ID NO: 115), 492F+573P+574G+580K (e.g. SEQID NO: 238), 492G+573P+574G+580K (e.g. SEQ ID NO: 234),492D+573P+574G+580K (e.g. SEQ ID NO: 235), 492F+573P+574H+580R (e.g. SEQID NO: 239), 492D+573P+574H+580K (e.g. SEQ ID NO: 264),492G+573P+574H+580R (e.g. SEQ ID NO: 244), 492D+573P+574H+580R (e.g. SEQID NO: 236) or 492F+573P+574H+580K (e.g. SEQ ID NO: 237).

A variant albumin may comprise alterations at positions corresponding topositions 550+573+574+580 of SEQ ID NO: 2 (or equivalent position ofother albumins or variants or fragments thereof). Such alterations maycomprise 550K+573P+574H+580K (e.g. SEQ ID NO: 265).

A variant albumin may comprise alterations at positions corresponding topositions 492+550+573+574+580 of SEQ ID NO: 2 (or equivalent position ofother albumins or variants or fragments thereof). Such alterations maycomprise 492D+550K+573P+574H+580K (e.g. SEQ ID NO: 266) or492G+550K+573P+574H+580K (e.g. SEQ ID NO: 267). Such alterations maycomprise 492D+550K+573P+574H (e.g. SEQ ID NO: 256).

Particularly preferred variants include:

a variant albumin with alterations at positions corresponding topositions 492 and 580 in SEQ ID NO: 2, such as (i) E492G and Q580K, or(ii) E492D and Q580K (or equivalent positions of other albumins orvariants or fragment thereof);

a variant albumin comprising alterations at positions corresponding topositions 492 and 574 in SEQ ID NO: 2, such as (i) E492G and K574H, (ii)E492D and K574H, (iii) E492D and K574K, or (iv) E492G and K574K (orequivalent positions of other albumins or variants or fragment thereof);

a variant albumin comprising alterations at positions corresponding topositions 492 and 550 in SEQ ID NO: 2, such as (i) E492G and D550K, or(ii) E492D and D550K (or equivalent positions of other albumins orvariants or fragment thereof);

a variant albumin comprising alterations at positions corresponding topositions 550 and 573 in SEQ ID NO: 2, such as (i) D550K and K573P, (ii)D550L and K573P or (iii) D550M and K573P (or equivalent positions ofother albumins or variants or fragment thereof);

a variant albumin comprising alterations at positions corresponding topositions 550 and 574 in SEQ ID NO: 2, such as (i) D550K and K574H, or(ii) D550L and K574H (or equivalent positions of other albumins orvariants or fragment thereof);

a variant albumin comprising alterations at positions corresponding topositions 550 and 580 in SEQ ID NO: 2, such as (i) D550M and Q580K, (ii)D550L and Q580K or (iii) D550K and Q580K (or equivalent positions ofother albumins or variants or fragment thereof);

a variant albumin with alterations (e.g. comprising alterations) atpositions corresponding to positions 580 and 573 in SEQ ID NO: 2, suchas Q580K and K573P (or equivalent positions of other albumins orvariants or fragment thereof) and preferably one or more (several) otheralterations such at a position selected from 492, 550 and 574. If thereis a K at position 580 and a P at position 573 it is preferred that thepolypeptide comprises an additional alteration at a position selectedfrom the group consisting of 492, 550, and 574;

a variant albumin with alterations (e.g. comprising alterations) atpositions corresponding to positions 492 and 573 in SEQ ID NO: 2, suchas:

a variant albumin comprising alterations at positions corresponding topositions 492 and 573 in SEQ ID NO: 2, such as (i) E492D and K573P (orequivalent positions of other albumins or variants or fragment thereof)or (ii) E492G and K573P or (iii) E492G and K573A (or equivalentpositions of other albumins or variants or fragment thereof), andpreferably one or more (several) other alterations such at a positionselected from 550, 574 and 580. If there is a G at position 492 and an Aor P at position 573 it is preferred that the polypeptide comprises anadditional alteration at a position selected from the group consistingof 550, 574 and 580 (or equivalent positions of other albumins orvariants or fragment thereof);

a variant albumin comprising alterations at positions corresponding topositions 573 and 574 in SEQ ID NO: 2, such as K573P and K574H (orequivalent positions of other albumins or variants or fragment thereof,and preferably one or more (several) alterations such as at a positionselected from 492, 550 and 580. If there is a P at position 573 and an Nat position 574 it is preferred that the polypeptide comprises anadditional alteration at a position selected from the group consistingof 492, 550, and 580 (or equivalent positions of other albumins orvariants or fragment thereof);

a variant albumin comprising alterations at positions corresponding topositions 574 and 580 in SEQ ID NO: 2, such as 574H and 580K (orequivalent positions of other albumins or variants or fragment thereof,and preferably one or more (several) alterations such as at a positionselected from 492, 550 and 573. If there is a N at position 574 and a Kat position 580 it is preferred that the polypeptide comprises anadditional alteration at a position selected from the group consistingof 492, 550, and 573 (or equivalent positions of other albumins orvariants or fragment thereof);

a variant albumin comprising alterations at positions corresponding topositions 492, 550 and 573 in SEQ ID NO: 2, such as E492G, D550K andK573P e.g. SEQ ID NO: 254 or such as E492D, D550K and K573P e.g. SEQ IDNO: 253;

a variant albumin comprising alterations at positions corresponding topositions 550, 573 and 580 in SEQ ID NO: 2, such as D550K, K573P andQ580K e.g. SEQ ID NO: 133;

a variant albumin comprising alterations at positions corresponding topositions 550, 573 and 580 in SEQ ID NO: 2, such as D550L, K573P andQ580K e.g. SEQ ID NO:248 or such as D550M, K573P and Q580K e.g. SEQ IDNO:252;

a variant albumin comprising alterations at positions corresponding topositions 550, 573 and 574 in SEQ ID NO: 2, such as D550L, K573P andK574H e.g. SEQ ID NO:246;

a variant albumin comprising alterations at positions corresponding topositions 550, 573 and 574 in SEQ ID NO: 2, such as D550K, K573P andK574H e.g. SEQ ID NO: 131;

a variant albumin comprising alterations at positions corresponding topositions 492, 573 and 580 in SEQ ID NO: 2, such as E492G, K573P andQ580K e.g. SEQ ID NO: 242;

a variant albumin comprising alterations at positions corresponding topositions 492, 573 and 580 in SEQ ID NO: 2, such as E492D, K573P andQ580K e.g. SEQ ID NO: 234;

a variant albumin comprising alterations at positions corresponding topositions 492, 550, 573 and 574 in SEQ ID NO: 2, such as E492D, D550K,K573P and K574H e.g. SEQ ID NO: 256;

a variant albumin comprising alterations at positions corresponding topositions 492, 550, 573 and 574 in SEQ ID NO: 2, such as E492G, D550K,K573P and K574H e.g. SEQ ID NO: 257;

a variant albumin comprising alterations at positions corresponding topositions 573, 574 and 580 in SEQ ID NO: 2, such as K573P, K574H andQ580K e.g. SEQ ID NO: 135;

a variant albumin with alterations (e.g. comprising alterations) atpositions corresponding to positions 492, 573, 574 and 580 in SEQ ID NO:2, such as E492D, K573P, K574H and Q580K (or equivalent positions ofother albumins or variants or fragment thereof), e.g. SEQ ID NO: 114;

a variant albumin with alterations (e.g. comprising alterations) atpositions corresponding to positions 492, 573, 574 and 580 in SEQ ID NO:2, such as E492G, K573P, K574H and Q580K (or equivalent positions ofother albumins or variants or fragment thereof), e.g. SEQ ID NO: 115.

It is preferred that the variant albumin does not consist of SEQ ID NO:2 with only the following alterations:K573P+K574N+A577T+A578R+S579C+Q580K+A581 D+G584A;K573P+A577E+A578S+Q580K+A582T; E492G+K573A; E492G+N503K+K573A;E492G+N503H+K573A; E492G+K573P; E492G+N503K+K573P; E492G+N503H+K573P.Such variant albumins are disclosed in WO2011/051489.

It is preferred that the variant albumin does not consist of SEQ ID NO:2 with amino acids 573 to 585 replaced with (a) KKLVAASQAALGL, (b)PKFVAASQAALA, (c) PNLVTRCKDALA, (d) PKLVESSKATLG or (e) PKLVASTQAALA.

It is preferred that the variant albumin, a fragment thereof or fusionpolypeptide comprising the variant albumin or fragment thereof hasaltered binding affinity to FcRn and/or an altered plasma half-lifecompared with the corresponding parent or reference albumin, fragmentthereof, or fusion polypeptide comprising the variant albumin orfragment thereof and/or an altered binding affinity to FcRn.

In a particularly preferred embodiment the parent or reference albuminis HSA (SEQ ID NO: 2) and the variant albumin, a fragment thereof orfusion polypeptide comprising the variant albumin or fragment thereofhas altered binding affinity to FcRn and/or an altered plasma half-lifecompared with the HSA, the corresponding fragment or fusion polypeptidecomprising HSA or fragment thereof and/or an altered binding affinity toFcRn.

The correlation between binding of albumin to its receptor and plasmahalf-life has been realized by the present inventors based on thenatural occurring allele of HSA D494N. The inventors have previouslyanalyzed this allele and found that it has a lower affinity to itsreceptor FcRn than the affinity of WT HSA to FcRn.

Further, it has been disclosed that a transgenic mouse having thenatural mouse FcRn replaced with human FcRn has a higher serum albuminlevel than normal mouse (J Exp Med. (2003) 197(3):315-22). It haspreviously been discovered that human FcRn has a higher affinity tomouse serum albumin than mouse FcRn has to mouse serum albumin and,therefore, the observed increase in serum albumin in the transgenic micecorresponds with a higher affinity between serum albumin and itsreceptor, confirming the correlation between albumin binding to FcRn andplasma half-life. In addition, variants of albumin that have little orno binding to FcRn have been shown to have reduced half-life in a mousemodel, Kenanova et al (2009) J. Nucl. Med.; 50 (Supplement 2):1582).

One way to determine whether the affinity of a variant albumin to FcRnis higher or lower than the parent or reference albumin is to use theSurface Plasmon Resonance assay (SPR) as described below. The skilledperson will understand that other methods might be useful to determinewhether the affinity of a variant albumin to FcRn is higher or lowerthan the affinity of the parent or reference albumin to FcRn, e.g.,determination and comparison of the binding constants KD. The bindingaffinity (KD) between a first molecule (e.g. ligand) and a secondmolecule (e.g. receptor) is a function of the kinetic constants forassociation (on rate, k_(a)) and dissociation (off-rate, k_(d))according to KD=k_(d)/k_(a). Thus, according to the invention variantalbumins having a KD that is lower than the KD for natural HSA isconsidered to have a higher plasma half-life than HSA and variantalbumins having a KD that is higher than the KD for natural HSA isconsidered to have a lower plasma half-life than HSA.

In an embodiment of the invention, the variants of albumin or fragmentsthereof, or fusion polypeptides comprising variant albumin or a fragmentthereof according to the invention have a plasma half-life that islonger than the plasma half-life of the parent or reference albuminfragment thereof or fusion polypeptide comprising the parent orreference albumin or a fragment thereof and/or an stronger bindingaffinity to FcRn.

In a further embodiment the variants of albumin or fragments thereof, orfusion polypeptides comprising variant albumin or fragments thereofaccording to the invention have a plasma half-life that is shorter thanthe plasma half-life of the parent or reference albumin fragment thereofor fusion polypeptide comprising the parent or reference albumin or afragment thereof and/or an weaker binding affinity to FcRn.

In addition to alterations at two or more positions selected from 492,550, 573, 574 and/or 580 (or equivalent position of other albumins orvariants or fragments thereof) the variant albumin or fragments thereof,or fusion polypeptides comprising variant albumin or fragments thereofaccording to the invention may contain additional substitutions,deletions or insertions in other positions of the molecules. Suchadditional substitutions, deletions or insertions may be useful in orderto alter other properties of the molecules such as but not limited toaltered glycosylation; introduction of reactive groups of the surfacesuch a thiol groups, removing/generating a carbamoylation site; etc.

Residues that might be altered in order to provide reactive residues onthe surface and which advantageously could be applied to the inventionhas been disclosed in WO2010/092135 (incorporated herein by reference).Particular preferred residues include the positions corresponding topositions in SEQ ID NO: 2.

As examples of alterations that can be made in SEQ ID NO: 2 or incorresponding positions in other albumins in order to provide a reactivethiol group on the surface includes alterations corresponding tofollowing alterations in SEQ ID NO: 2: L585C, D1C, A2C, D562C, A364C,A504C, E505C, T79C, E86C, D129C, D549C, A581C, D121C, E82C, S270C,A578C, L595LC, D1 DC, A2AC, D562DC, A364AC, A504AC, E505EC, T79TC,E86EC, D129DC, D549DC, A581AC, A581AC, D121DC, E82EC, S270SC, A579AC,C360*, C316*, C75*, C168*, C558*, C361*, C91*, C124*, C169* and C567*.Alternatively a cysteine residue may be added to the N or C terminal ofalbumin. The term ‘reactive thiol’ means and/or includes a thiol groupprovided by a Cys which is not disulphide bonded to a Cysteine and/orwhich is sterically available for binding to a partner such as aconjugation partner.

Fusion Polypeptides

A second aspect of the invention relates to fusion polypeptides.Therefore, the variants of albumin or fragments thereof according to theinvention may be fused with a non-albumin polypeptide fusion partner.The fusion partner may in principle be any polypeptide but generally itis preferred that the fusion partner is a polypeptide havingtherapeutic, prophylactic (including vaccine), diagnostic, imaging orother beneficial properties. Such properties may be referred to as‘pharmaceutically beneficial properties’. Fusion polypeptides comprisingalbumin or fragments thereof are known in the art. It has been foundthat such fusion polypeptides comprising albumin or a fragment thereofand a fusion partner polypeptide have a longer plasma half-life comparedto the unfused fusion partner polypeptide alone. According to theinvention it is possible to alter the plasma half-life of the fusionpolypeptides according to the invention compared to the correspondingfusion polypeptides of the prior art. ‘Alter’ includes both increasingthe plasma half-life or decreasing the plasma half-life. Increasing theplasma half-life is preferred. The invention allows tailoring ofhalf-life to a term desired.

One or more (several) therapeutic, prophylactic (including vaccine),diagnostic, imaging or other beneficial polypeptides may be fused to theN-terminus, the C-terminus of albumin, inserted into a loop in thealbumin structure or any combination thereof. It may or it may notcomprise linker sequences separating the various components of thefusion polypeptide.

Teachings relating to fusions of albumin or a fragment thereof are knownin the art and the skilled person will appreciate that such teachingscan also be applied to the invention. WO 2001/79271A (particularly page9 and/or Table 1), WO 2003/59934 (particularly Table 1), WO03/060071(particularly Table 1) and WO01/079480 (particularly Table 1) (eachincorporated herein by reference in their entirety) also containexamples of therapeutic, prophylactic (including vaccine), diagnostic,imaging or other beneficial polypeptides that may be fused to albumin orfragments thereof, and these examples apply also to the invention.

Further preferences for the second aspect of the invention include thoseof the first aspect of the invention and those provided below thetwelfth aspect of the invention. The skilled person understands that anyaspect of the invention may be combined with another aspect or aspectsof the invention and/or with one or more (several) of the preferencesfor the aspects of the invention and/or other disclosures made herein.

Polynucleotides

A third aspect of the invention relates to isolated polynucleotides thatencode any of the variants or fusion polypeptides of the invention. Thepolynucleotide may be an isolated polynucleotide. The polynucleotide maybe comprised in a vector (such as a plasmid) and/or in a host cell.

Further preferences for the third aspect of the invention include thoseof the first aspect of the invention and those provided below thetwelfth aspect of the invention. The skilled person understands that anyaspect of the invention may be combined with another aspect or aspectsof the invention and/or with one or more (several) of the preferencesfor the aspects of the invention and/or other disclosures made herein.

Nucleic Acid Constructs

A fourth aspect of the invention relates to nucleic acid constructscomprising a polynucleotide encoding a variant or fusion polypeptide ofthe invention operably linked to one or more (several) control sequencesthat direct the expression of the coding sequence in a suitable hostcell under conditions compatible with the control sequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of a variant. Manipulation of the polynucleotide prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter sequence, which is recognized bya host cell for expression of the polynucleotide. The promoter sequencecontains transcriptional control sequences that mediate the expressionof the variant. The promoter may be any nucleic acid sequence that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaeprotease A (PRA1), Saccharomyces cerevisiae protease B (PRB1),Saccharomyces cerevisiae translation elongation factor (TEF1),Saccharomyces cerevisiae translation elongation factor (TEF2),Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI),Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomycescerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeasthost cells are described by Romanos et al., 1992, Yeast 8: 423-488.

The skilled person knows useful promoters for use in rice and mammaliancells, such as CHO or HEK. In a rice host, useful promoters are obtainedfrom cauliflower mosaic virus 35S RNA gene (CaMV35S), maize alcoholdehydrogenase (Adh1) and alpha Amy3.

In a mammalian host cell, such as CHO or HEK, useful promoters areobtained from Cytomegalovirus (CMV) and CAG hybrid promoter (hybrid ofCMV early enhancer element and chicken beta-actin promoter), Simianvacuolating virus 40 (SV40).

The control sequence may also be a suitable transcription terminatorsequence, which is recognized by a host cell to terminate transcription.The terminator sequence is operably linked to the 3′-terminus of thepolynucleotide encoding the variant. Any terminator that is functionalin the host cell may be used.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C(CYC1), Saccharomyces cerevisiae alcohol dehydrogenase(ADH1) and Saccharomyces cerevisiae glyceraldehyde-3-phosphatedehydrogenase. Other useful terminators for yeast host cells aredescribed by Romanos et al., 1992, supra. The skilled person knowsuseful terminators for use in rice and mammalian cells, such as CHO orHEK. For example, in a rice host, preferred terminators are obtainedfrom Agrobacterium tumefaciens nopaline synthase (Nos) and cauliflowermosaic virus 35S RNA gene (CaMV35S).

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′-terminus ofthe polynucleotide encoding the variant. Any leader sequence that isfunctional in the host cell may be used.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding region naturally linked in translation reading frame withthe segment of the coding region that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding region that is foreign to the coding sequence. Theforeign signal peptide coding region may be required where the codingsequence does not naturally contain a signal peptide coding region.Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to enhancesecretion of the variant. However, any signal peptide coding region thatdirects the expressed variant into the secretory pathway of a host cellmay be used.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra. The skilled person knows useful signalpeptides for use in rice and mammalian cells, such as CHO or HEK.

Where both signal peptide and propeptide regions are present at theN-terminus of a variant, the propeptide region is positioned next to theN-terminus of the variant and the signal peptide region is positionednext to the N-terminus of the propeptide region.

Further preferences for the fourth aspect of the invention include thoseof the first aspect of the invention and those provided below thetwelfth aspect of the invention. The skilled person understands that anyaspect of the invention may be combined with another aspect or aspectsof the invention and/or with one or more (several) of the preferencesfor the aspects of the invention and/or other disclosures made herein.

Preparation of Variants

A fifth aspect of the invention relates to a method for preparing orobtaining a variant albumin or fragment thereof, or fusion polypeptidescomprising the variant albumin or fragments thereof, or associates ofvariant albumin or fragment thereof comprising:

(a) introducing into a parent albumin or fragments thereof, or fusionpolypeptides comprising the parent albumin or fragments thereof analteration at two or more positions selected from 492, 550, 573, 574 and580, preferably as described for the first aspect of the invention; and

(b) recovering the variant albumin or fragment thereof, or fusionpolypeptides comprising the variant albumin or fragment thereof.

Preferred alterations are as described in relation to the first aspectof the invention. The resultant variant albumin or fragment thereof mayhave altered FcRn-binding affinity compared to the FcRn-binding affinityof a reference such as a parent albumin or fragment which does notcomprise the alterations. More preferably, the resultant variant albuminor fragment thereof has a stronger FcRn-binding affinity.

The invention includes a method for preparing a polypeptide which is avariant of albumin, fragment thereof or fusion polypeptide comprisingsaid variant albumin or fragment thereof having a binding affinity toFcRn which is altered compared to the binding affinity of a referencealbumin, fragment or fusion thereof to FcRn, comprising:

(a) providing a nucleic acid encoding a parent albumin such as analbumin having at least 60% sequence identity to SEQ ID NO: 2;

(b) modifying the sequence of step (a), to encode a polypeptide which isa variant albumin, fragment thereof or fusion polypeptide comprisingsaid variant albumin or fragment thereof comprising alterations at twoor more positions corresponding to positions selected from selectedamong two or more of the group consisting of positions 492, 550, 573,574 and 580 in SEQ ID NO: 2;

(c) optionally, introducing the modified sequence of step (b) in asuitable host cell;

(d) optionally, growing the cells in a suitable growth medium undercondition leading to expression of the polypeptide; and

(e) optionally, recovering the polypeptide from the growth medium;

wherein the polypeptide has an altered binding affinity to FcRn and/oran altered plasma half-life compared with the half-life of a parentalbumin, reference albumin, fragment thereof or fusion polypeptidecomprising said parent albumin, reference albumin or fragment or fusionthereof.

It is preferred that the parent albumin and/or the variant albumincomprises or consists of:

(a) a polypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 2;

(b) a polypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, or (ii) the full-length complement of (i);

(c) a polypeptide encoded by a polynucleotide having at least 60%identity to the polypeptide coding sequence of SEQ ID NO: 1; and/or

(d) a fragment of the mature polypeptide of SEQ ID NO: 2.

The variants can be prepared by those skilled persons using anymutagenesis procedure known in the art, such as site-directedmutagenesis, synthetic gene construction, semi-synthetic geneconstruction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (several)mutations (alterations) are created at one or more (several) definedsites in a polynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests at theplasmid and the oligonucleotide is the same, permitting ligation of theplasmid and insert to one another. See, e.g., Scherer and Davis, 1979,Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton et al., 1990,Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication NO:2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the invention.There are many commercial kits available that can be used to preparevariants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide sub sequences may then be shuffled.

Further preferences for the fifth aspect of the invention include thoseof the first aspect of the invention and those provided below thetwelfth aspect of the invention. The skilled person understands that anyaspect of the invention may be combined with another aspect or aspectsof the invention and/or with one or more (several) of the preferencesfor the aspects of the invention and/or other disclosures made herein.

Methods of Production

A sixth aspect of the invention relates to methods of preparation of avariant according to the invention. The variants of the invention can beprepared using techniques well known to the skilled person. Oneconvenient way is by cloning nucleic acid encoding the parent albumin ora fragment thereof or fusion polypeptide comprising albumin or afragment thereof, modifying said nucleic acid to introduce the desiredsubstitution(s) at two or more positions corresponding to positionsselected from 492, 550, 573, 574 and 580 of the mature polypeptide ofSEQ ID NO: 2 (or equivalent positions in other albumins or fragmentsthereof), preferably as described for the first or fifth aspects of theinvention, preparing a suitable genetic construct where the modifiednucleic acid is placed in operative connection with suitable regulatorygenetic elements, such as promoter, terminator, activation sites,ribosome binding sites etc., introducing the genetic construct into asuitable host organism, culturing the transformed host organism underconditions leading to expression of the variant and recovering thevariant. All these techniques are known in the art and it is within theskills of the average practitioner to design a suitable method forpreparing a particular variant according to the invention.

The variant polypeptide of the invention may also be connected to asignal sequence in order to have the variant polypeptide secreted intothe growth medium during culturing of the transformed host organism. Itis generally advantageous to have the variant polypeptide secreted intothe growth medium in order to ease recovery and purification.

Techniques for preparing variant polypeptides have also been disclosedin WO 2009019314 (included by reference) and these techniques may alsobe applied to the invention.

Albumins have been successfully expressed as recombinant proteins in arange of hosts including fungi (including but not limited to Aspergillus(WO06066595), Kluyveromyces (Fleer 1991, Bio/technology 9, 968-975),Pichia (Kobayashi 1998 Therapeutic Apheresis 2, 257-262) andSaccharomyces (Sleep 1990, Bio/technology 8, 42-46)), bacteria(Pandjaitab 2000, J. Allergy Clin. Immunol. 105, 279-285)), animals(Barash 1993, Transgenic Research 2, 266-276) and plants (including butnot limited to potato and tobacco (Sijmons 1990, Bio/technology 8, 217and Farran 2002, Transgenic Research 11, 337-346) and rice e.g. Oryzasativa) and mammalian cells such as CHO and HEK. The variant polypeptideof the invention is preferably produced recombinantly in a suitable hostcell. In principle any host cell capable of producing a polypeptide insuitable amounts may be used and it is within the skills of the averagepractitioner to select a suitable host cell according to the invention.A preferred host organism is yeast, preferably selected amongSaccharomycacae, more preferred Saccharomyces cerevisiae.

The variant polypeptides of the invention may be recovered and purifiedfrom the growth medium using a combination of known separationtechniques such as filtration, centrifugation, chromatography, andaffinity separation techniques etc. It is within the skills of theaverage practitioner to purify the variants of the invention using aparticular combination of such known separation steps. As an example ofpurification techniques that may be applied to the variants of theinvention can be mentioned the teaching of WO00/44772.

The variant polypeptides of the invention may be used for delivering atherapeutically beneficial compound (including prophylacticallybeneficial compound such as a vaccine) to an animal or a humanindividual in need thereof. Such therapeutically beneficial compoundsinclude, but are not limited, to labels and readily detectable compoundsfor use in diagnostics, such as various imaging techniques;pharmaceutical active compounds such as drugs, or specifically bindingmoieties such as antibodies. The variants of the invention may even beconnected to two or more different therapeutically beneficial compounds,e.g., an antibody and a drug, which gives the combined molecule theability to bind specifically to a desired target and thereby provide ahigh concentration of the connected drug at that particular target.

Further preferences for the sixth aspect of the invention include thoseof the first aspect of the invention and those provided below thetwelfth aspect of the invention. The skilled person understands that anyaspect of the invention may be combined with another aspect or aspectsof the invention and/or with one or more (several) of the preferencesfor the aspects of the invention and/or other disclosures made herein.

Conjugates

A seventh aspect of the invention relates to conjugates (conjugations).Therefore, the variants of albumin or fragments thereof or fusionpolypeptides according to the invention may be conjugated to a secondmolecule (‘conjugation partner’) using techniques known within the art.The conjugation partner may be a therapeutic, prophylactic (includingvaccine), diagnostic, imaging or other beneficial moiety. Saidconjugation partner may be a polypeptide or a non-polypeptide chemical.The conjugation partner may be a polypeptide, chemical (e.g. chemicallysynthesised drug) or a nucleic acid (e.g. DNA, RNA, siRNA).

Said second molecule may comprise a diagnostic or imaging moiety, and inthis embodiment the conjugate may be useful as a diagnostic tool such asin imaging; or the second molecule may be a therapeutic or prophylactic(e.g. vaccine) compound and in this embodiment the conjugate may be usedfor therapeutic or prophylactic (e.g. vaccination) purposes where theconjugate will have the therapeutic or prophylactic properties of thetherapeutic or prophylactic compound as well as the desirable plasmahalf-life provided by the albumin part of the conjugate. Conjugates ofalbumin and a therapeutic molecule are known in the art and it has beenverified that such conjugates have long plasma half-life compared withthe non-conjugated, free therapeutic molecule as such. According to theinvention it is possible to alter the binding affinity to FcRn and/orplasma half-life of the conjugate according to the invention compared tothe corresponding conjugates of the prior art. ‘Alter’ includes bothincreasing the plasma half-life and decreasing the plasma half-lifebinding affinity to FcRn and/or increasing the binding affinity anddecreasing the binding affinity to FcRn. Increasing the plasma half-lifeand/or binding affinity to FcRn is preferred. The conjugates mayconveniently be linked via a free thiol group present on the surface ofHSA (amino acid residue 34 of mature HSA) using well known chemistry.

In one particular preferred aspect the variant albumin or fragmentthereof is conjugated to a beneficial therapeutic or prophylactic(including vaccine) compound and the conjugate is used for treatment ofa condition in a patient in need thereof, which condition is responsiveto the particular selected therapeutic compound. Techniques forconjugating such a therapeutically useful compound to the variantalbumin or fragment thereof are known in the art. WO 2009/019314(incorporated herein by reference in its entirety) discloses examples oftechniques suitable for conjugating a therapeutically compound to apolypeptide which techniques can also be applied to the invention.Further WO 2009/019314 discloses examples of compounds and moieties thatmay be conjugated to substituted transferrin and these examples may alsobe applied to the invention. The teaching of WO 2009/019314 is includedherein by reference.

HSA contains in its natural form one free thiol group (at Cys34) thatconveniently may be used for conjugation. As a particular embodimentwithin this aspect the variant albumin or fragment thereof may comprisefurther modifications provided to generate additional free thiol groupson the surface. This has the benefit that the payload of the variantalbumin or fragment thereof is increased so that more than one moleculeof the therapeutic (e.g. prophylactic) compound can be conjugated toeach molecule of variant albumin or fragment thereof, or two or more(several) different therapeutic compounds may be conjugated to eachmolecule of variant albumin or fragment thereof, e.g., a compound havingtargeting properties such as an antibody specific for example a tumour;and a cytotoxic drug conjugated to the variant albumin or fragmentthereof thereby creating a highly specific drug against a tumour.Teaching of particular residues that may be modified to provide forfurther free thiol groups on the surface can be found in co-pendingpatent application WO 2010/092135, which is incorporated by reference.

The conjugation partner may alternatively be conjugated to a fusionpolypeptide (described herein), resulting in a molecule comprising afusion partner fused to the albumin as well as a conjugation partnerconjugated to the same albumin or even to the fusion partner.

Further preferences for the seventh aspect of the invention includethose of the first aspect of the invention and those provided below thetwelfth aspect of the invention. The skilled person understands that anyaspect of the invention may be combined with another aspect or aspectsof the invention and/or with one or more (several) of the preferencesfor the aspects of the invention and/or other disclosures made herein.

Associates

An eighth aspect of the invention relates to associates. Therefore, thevariants of albumin or fragments thereof or fusion polypeptides mayfurther be used in form of “associates”. In this connection the term“associate” is intended to mean a compound comprising a variant ofalbumin or a fragment thereof and another compound bound or associatedto the variant albumin or fragment thereof by non-covalent binding. Asan example of such an associate can be mentioned an associate consistingof variant albumin and a lipid associated to albumin by a hydrophobicinteraction. Such associates are known in the art and they may beprepared using well known techniques. As an example of a preferredassociate according to the invention can be mentioned, an associatecomprising variant albumin and a taxane, a taxol or taxol derivative(e.g. paclitaxel). Further examples of associates comprise atherapeutic, prophylactic (including vaccine), diagnostic, imaging orother beneficial moiety.

The half-life of an albumin associate according to the invention may belonger or shorter than the half-life of the ‘other compound’ alone. Thehalf-life of an albumin associate according to the invention may belonger or shorter than the half-life of the analogous/equivalent albuminassociate comprising or consisting of a reference albumin such as nativeHSA (instead of an albumin variant or derivative according to theinvention) and the ‘other compound’. Likewise, the binding affinity toFcRn of an albumin associate according to the invention may be strongeror weaker than the binding affinity to FcRn of the analogous/equivalentalbumin associate comprising or consisting of a reference albumin suchas native HSA (instead of an albumin variant or derivative according tothe invention) and the ‘other compound’. Methods for the preparation ofassociates are well-known to the skilled person, for example,formulation (by association) of HSA with Lipo-compounds is described inHussain, R. and Siligardi, G. (2006) International Journal of PeptideResearch and Therapeutics, Vol. 12, NO: 3, pp. 311-315.

Further preferences for the eighth aspect of the invention include thoseof the first aspect of the invention and those provided below thetwelfth aspect of the invention. The skilled person understands that anyaspect of the invention may be combined with another aspect or aspectsof the invention and/or with one or more (several) of the preferencesfor the aspects of the invention and/or other disclosures made herein.

Compositions

A ninth aspect of the invention relates to compositions. Therefore theinvention is also directed to the use of a variant of albumin or afragment thereof or fusion polypeptides comprising variant albumin orfragments thereof, or a conjugate comprising a variant of albumin or afragment thereof, or an associate comprising a variant of albumin or afragment thereof for the manufacture of a pharmaceutical composition,wherein the variant of albumin or a fragment thereof or fusionpolypeptides comprising variant albumin or fragments thereof, or aconjugate comprising a variant of albumin or a fragment thereof, or anassociate comprising a variant of albumin or a fragment thereof has analtered binding affinity to FcRn and/or an altered plasma half-lifecompared with HSA or the corresponding fragment thereof or fusionpolypeptide comprising HSA or fragment thereof or conjugate comprisingHSA.

In this connection the corresponding fragment of HSA is intended to meana fragment of HSA that aligns with and has same number of amino acids asthe fragment of the variant albumin with which it is compared. Similarlythe corresponding fusion polypeptide comprising HSA or conjugatecomprising HSA is intended to mean molecules having same size and aminoacid sequence as the fusion polypeptide of conjugate comprising variantalbumin, with which it is compared.

The composition may comprise a pharmaceutically acceptable carrier orexcipient such as water, polysorbate 80 or those specified in the USPharmacopoeia for human albumin.

Further preferences for the ninth aspect of the invention include thoseof the first aspect of the invention and those provided below thetwelfth aspect of the invention. The skilled person understands that anyaspect of the invention may be combined with another aspect or aspectsof the invention and/or with one or more (several) of the preferencesfor the aspects of the invention and/or other disclosures made herein.

Nanoparticles

A tenth aspect of the invention relates to a nanoparticle comprising avariant, fusion, conjugate, associate, nanoparticle, composition orpolynucleotide as disclosed herein.

Techniques for incorporation of a molecule into nano- or microparticlesare known in the art. Preferred methods for preparing nano- ormicroparticles that may be applied to the albumin, variant, fragment,fusion, conjugate or associate thereof according to the invention isdisclosed in WO 2004/071536 or WO2008/007146 or Oner & Groves(Pharmaceutical Research, Vol 10(9), 1993, pages 1387 to 1388) which areincorporated herein by reference. Preferably the average diameter of anano-particle is from 5 to 1000 nm, more preferably 5, 10, 20, 30, 40,50, 80, 100, 130, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 999 to5, 10, 20, 30, 40, 50, 80, 100, 130, 150, 200, 300, 400, 500, 600, 700,800, 900, or 1000 nm. An advantage of a microparticle less than 200 nmdiameter, and more particularly less than 130 nm, is that is amenable tosterilisation by filtration through a 0.2 μm (micron) filter.Preferably, the average diameter of a micro-particle is from 1000 nm (1μm (micron)) to 100 μm (micron), more preferably from 1, 2, 5, 10, 20,30, 40, 50, 60, 70, 80, 90, 100 to 1, 2, 5, 10, 20, 30, 40, 50, 60, 70,80, 90, 100 μm (micron).

Further preferences for the tenth aspect of the invention include thoseof the first aspect of the invention and those provided below thetwelfth aspect of the invention. The skilled person understands that anyaspect of the invention may be combined with another aspect or aspectsof the invention and/or with one or more (several) of the preferencesfor the aspects of the invention and/or other disclosures made herein.

Uses

An eleventh aspect of the invention relates to use of a variant albumin,fragment, fusion or conjugate thereof or nanoparticle or associatethereof. Use may be, for example, in a method of treatment, prophylaxis,diagnosis or imaging. The variant albumin or fragments thereof or fusionpolypeptides comprising variant albumin or fragments thereof accordingto the invention have the benefit that their binding affinity to FcRnand/or plasma half-life is altered compared to the parent or referencealbumin or fragments thereof or fusion polypeptides comprising parent orreference albumin or fragments thereof. This has the advantage that thebinding affinity to FcRn and/or plasma half-life of conjugatescomprising variant albumin or a fragment thereof or fusion polypeptidecomprising variant albumin or a fragment thereof, or an associatecomprising variant albumin or a fragment thereof according to theinvention can be selected in accordance with the particular therapeuticpurpose.

In some situations, it would be advantageous to use an albumin, variant,fragment, fusion, conjugate or associate or composition thereof having alonger plasma half-life than the reference molecule or composition sincethis would have the benefit that the administration of the albumin,variant, fragment, fusion, conjugate or associate or composition thereofwould be needed less frequently or at a reduced dose (and consequentlywith fewer side effects) compared to the situation where the referencemolecule or composition was used. With respect to the use of a variant,fusion, conjugate, associate, nanoparticle, composition orpolynucleotide the albumin moiety may comprise one more alterations asdisclosed herein.

In other situations, it would be advantageous to use an albumin,variant, fragment, fusion, conjugate or associate or composition thereofhaving a shorter plasma half-life than the reference molecule orcomposition since this would have the benefit that the administration ofthe albumin, variant, fragment, fusion, conjugate or associate orcomposition thereof can be carried out at a higher dose compared to thesituation where the reference molecule or composition was used with thebenefit that the administered compound clears from the recipient morequickly than if the reference molecule or composition was used. Withrespect to the use of a variant, fusion, conjugate, associate,nanoparticle, composition or polynucleotide the albumin moiety maycomprise one more alterations as disclosed herein.

For example for a conjugate, associate or fusion polypeptide used forimaging purposes in animals or human beings, where the imaging moietyhas an very short half-life and a conjugate or a fusion polypeptidecomprising HSA has a plasma half-life that is far longer than needed forthe imaging purposes it would be advantageous to use a variant albuminor fragment thereof of the invention having a shorter plasma half-lifethan the parent or reference albumin or fragment thereof, to provideconjugates of fusion polypeptides having a plasma half-life that issufficiently long for the imaging purpose but sufficiently short to becleared form the body of the particular patient on which it is applied.

In another example for a conjugate, an associate or fusion polypeptidecomprising a therapeutic compound effective to treat or alleviate aparticular condition in a patient in need for such a treatment it wouldbe advantageous to use the variant albumin or fragment thereof having alonger plasma half-life than the parent or reference albumin or fragmentthereof, to provide associates or conjugates or fusion polypeptideshaving longer plasma half-lives which would have the benefit that theadministration of the associate or conjugate or fusion polypeptide ofthe invention would be needed less frequently or at reduced dose withless side effects compared to the situation where the parent orreference albumin or associates thereof or fragment thereof was used.For example, the invention provides a method of treating a proliferativedisease in an individual, comprising administering the individual aneffective amount of an associate according to the invention in which theassociate comprises a taxane, a taxol or taxol derivative (e.g.paclitaxel).

In a further aspect the invention relates to compositions comprising thevariant albumin, associates thereof or fragment thereof, variant albuminfragment or associates thereof or fusion polypeptide comprising variantalbumin or fragment thereof according to the invention. The compositionsare preferably pharmaceutical compositions. The composition may beprepared using techniques known in the area such as disclosed inrecognized handbooks within the pharmaceutical field. Since the albumin,variant, fragment, fusion, conjugate or associate thereof has a bindingaffinity to FcRn and/or plasma half-life which is modulated (i.e.stronger or weaker and/or longer or shorter) than that of a referencemolecule, the composition also has a binding affinity to FcRn and/ormodulated plasma half-life relative to an equivalent compositioncomprising the reference molecule in place of the albumin, variant,fragment, fusion, conjugate or associate thereof as described herein.The composition may be a vaccine. The polypeptide according to theinvention may be an active pharmaceutical or an excipient. Optionally,the composition is provided in unit dosage form.

Preferably the albumin, variant, fragment, fusion, conjugate orassociate thereof has a plasma half-life that is longer than the plasmahalf-life of the reference molecule e.g. the same composition exceptthat the albumin component (e.g. albumin, variant, fragment, fusion,conjugate or associate) is wild-type albumin (e.g. HSA) or a variant,fragment, fusion, conjugate or associate.

In a particular embodiment the compositions comprise a variant albuminor a fragment thereof according to the invention and a compoundcomprising a pharmaceutically beneficial moiety and an albumin bindingdomain (ABD). According to the invention ABD means a site, moiety ordomain capable of binding to circulating albumin in vivo and therebyconferring transport in the circulation of the ABD and any compound ormoiety bound to said ABD. ABD's are known in the art and have been shownto bind very tight to albumin so a compound comprising an ABD bound toalbumin will to a certain extent behave as a single molecule. Theinventors have realized by using the variant albumin or fragment thereofaccording to the invention together with a compound comprising apharmaceutically beneficial moiety and an ABD makes it possible to alterthe binding affinity to FcRn and/or plasma half-life of the compoundcomprising a pharmaceutically beneficial moiety and an ABD compared tothe situation where said compound were injected as such in a patienthaving need thereof or administered in a formulation comprising naturalalbumin or a fragment thereof.

The variant albumin or fragments thereof, conjugates comprising variantalbumin or a fragment thereof or fusion polypeptide comprising variantalbumin or a fragment thereof, or an associate comprising variantalbumin or a fragment thereof according to the invention may also beincorporated into nano- or microparticles using techniques well knownwithin the art. A preferred method for preparing nano- or microparticlesthat may be applied to the variant albumins or fragments thereofaccording to the invention is disclosed in WO 2004/071536 orWO2008/007146 or Oner & Groves (Pharmaceutical Research, Vol 10(9),1993, pages 1387 to 1388) which are incorporated herein by reference.

Further preferences for the eleventh aspect of the invention includethose of the first aspect of the invention and those provided below thetwelfth aspect of the invention. The skilled person understands that anyaspect of the invention may be combined with another aspect or aspectsof the invention and/or with one or more (several) of the preferencesfor the aspects of the invention and/or other disclosures made herein.

Method for Altering the FcRn-Binding Affinity or Half-Life of a Molecule

A twelfth aspect of the invention provides a method for altering theFcRn-binding affinity or half-life of a molecule comprising:

(a) where the molecule is a polypeptide, fusing or conjugating themolecule to a polypeptide disclosed herein or to a conjugate disclosedherein; associating the molecule to a polypeptide disclosed herein or toa conjugate disclosed herein; incorporating the molecule in ananoparticle disclosed herein or a composition disclosed herein;

(b) where the molecule is not a polypeptide, conjugating the molecule toa polypeptide disclosed herein or to a conjugate disclosed herein;associating the molecule to a polypeptide disclosed herein or to aconjugate a disclosed herein; incorporating the molecule in ananoparticle disclosed herein or a composition disclosed herein.

Examples of ‘molecule’ include those useful in therapy, prophylaxis(including those used in vaccines either as an active pharmaceuticalingredient or as an excipient), imaging and diagnosis, such as thosedescribed herein.

Further preferences for the twelfth aspect of the invention includethose of the first aspect of the invention and those provided below thistwelfth aspect of the invention. The skilled person understands that anyaspect of the invention may be combined with another aspect or aspectsof the invention and/or with one or more (several) of the preferencesfor the aspects of the invention and/or other disclosures made herein.

Preferences for all aspects of the invention are provided below. Theskilled person understands that any aspect of the invention may becombined with another aspect or aspects of the invention and/or with oneor more (several) of the preferences for the aspects of the inventionand/or other disclosures made herein.

The variant of albumin or a fragment thereof or fusion polypeptidescomprising variant albumin or fragments thereof, fragment thereof,conjugate, nanoparticle, associate or composition may have a plasmahalf-life that is either longer or shorter, preferably longer, than theplasma half-life than a corresponding albumin or a fragment thereof orfusion polypeptides comprising albumin or fragments thereof, fragmentthereof, conjugate, nanoparticle, associate or composition or a bindingto FcRn that is stronger or weaker, preferably weaker. Preferably thevariant of albumin or a fragment thereof or fusion polypeptidescomprising variant albumin or fragments thereof, fragment thereof,conjugate, nanoparticle, associate or composition has a plasma half-lifethat is longer than the plasma half-life of HSA or the correspondingalbumin or a fragment thereof or fusion polypeptides comprising albuminor fragments thereof, fragment thereof, conjugate, nanoparticle,associate or composition.

Alternatively, this may be expressed as the variant of albumin or afragment thereof or fusion polypeptides comprising variant albumin orfragments thereof, fragment thereof, conjugate, nanoparticle, associateor composition having a KD to FcRn (e.g. shFcRn) that is lower than thecorresponding KD for HSA to FcRn or the corresponding fragment thereofor fusion polypeptide comprising HSA or fragment thereof. Preferably,the KD for the variant of albumin or a fragment thereof or fusionpolypeptides comprising variant albumin or fragments thereof, fragmentthereof, conjugate, nanoparticle, associate or composition is less than0.9×KD for HSA to FcRn, more preferred less than 0.5×KD for HSA to FcRn,more preferred less than 0.1×KD for HSA to FcRn, even more preferredless than 0.05×KD for HSA to FcRn, even more preferred less than 0.02×KDfor HSA to FcRn and most preferred less than 0.01×KD for HSA to FcRn(where × means ‘multiplied by’). The KD of the variant of albumin or afragment thereof or fusion polypeptides comprising variant albumin orfragments thereof, fragment thereof, conjugate, nanoparticle, associateor composition may be between the KD of WT albumin (e.g. SEQ ID No. 2)for FcRn and the KD of HSA K573P (SEQ ID No. 3) for FcRn. Such KDsrepresent binding affinities that are higher than the binding affinitybetween HSA and FcRn. A higher binding affinity indicates a longerhalf-life, for example plasma half-life.

Alternatively, the variant of albumin or a fragment thereof or fusionpolypeptides comprising variant albumin or fragments thereof, fragmentthereof, conjugate, nanoparticle, associate or composition has a plasmahalf-life that is shorter than the plasma half-life of HSA or thecorresponding fragment thereof or fusion polypeptide comprising HSA orfragment thereof.

This may be expressed as the variant of albumin or a fragment thereof orfusion polypeptides comprising variant albumin or fragments thereof,fragment thereof, conjugate, nanoparticle, associate or compositionhaving a KD to FcRn that is higher than the corresponding KD for HSA toFcRn or the corresponding of albumin or a fragment thereof or fusionpolypeptides comprising albumin or fragments thereof, fragment thereof,conjugate, nanoparticle, associate or composition. Preferably, the KDfor the variant of albumin or a fragment thereof or fusion polypeptidescomprising variant albumin or fragments thereof, fragment thereof, or aconjugate comprising a variant of albumin or a fragment thereof is morethan 2×KD for HSA to FcRn, more preferred more than 5×KD for HSA toFcRn, more preferred more than 10×KD for HSA to FcRn, even morepreferred more than 25×KD for HSA to FcRn, most preferred more than50×KD, more than 60×, more than 70×KD, more than 80×, more than 90× ormore than 100×KD for HSA to FcRn. The variant of albumin or a fragmentthereof or fusion polypeptides comprising variant albumin or fragmentsthereof, fragment thereof, conjugate, nanoparticle, associate orcomposition may be a null binder to FcRn.

The variant of albumin or a fragment thereof or fusion polypeptidescomprising variant albumin or fragments thereof, fragment thereof, or aconjugate or nanoparticle or associate or composition comprising avariant of albumin or a fragment thereof is preferably the variant ofalbumin or a fragment thereof or fusion polypeptides comprising variantalbumin or fragments thereof, fragment thereof, or a conjugate ornanoparticle or associate or composition comprising a variant of albuminor a fragment thereof according to the invention. A lower bindingaffinity indicates a shorter half-life, for example plasma half-life.

One advantage of the invention is that it allows the half-life ofalbumin, a variant of albumin or a fragment thereof or fusionpolypeptides comprising variant albumin or fragments thereof, fragmentthereof, conjugate, nanoparticle, associate or composition to betailored in order to achieve a binding affinity or half-life which meetsthe needs of the user.

When determining and/or comparing KD, one or more (and preferably all)of the following parameters may be used:

Instrument: Biacore 3000 instrument (GE Healthcare)

Flow cell: CM5 sensor chip

FcRn: human FcRn, preferably soluble human FcRn, optionally coupled to atag such as

GST or His, most preferably His such as 6 histidines at the C-terminusof the beta-2-microglobulin (SEQ ID NO: 31).

Quantity of FcRn: 1200-2500 RU

Coupling chemistry: amine coupling chemistry (e.g. as described in theprotocol provided by the manufacturer of the instrument).

Coupling method: The coupling may be performed by injecting 20 μg/ml ofthe protein in 10 mM sodium acetate pH 5.0 (GE Healthcare). Phosphatebuffer (67 mM phosphate buffer, 0.15 M NaCl, 0.005% Tween 20) at pH 5.5)may be used as running buffer and dilution buffer. Regeneration of thesurfaces may be done using injections of HBS-EP buffer (0.01 M HEPES,0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20) at pH 7.4 (Biacore AB).

Quantity of injection of test molecule (e.g. HSA or variant) 20-0.032 μM

Flow rate of injection: constant, e.g. 30 μl/ml

Temperature of injection: 25° C.

Data evaluation software: BIAevaluation 4.1 software (BIAcore AB).

The preferred method for determining KD is provided in Example 2.

The invention discloses that two or more positions selected among thegroup consisting of positions 492, 550, 573, 574 and 580 in SEQ ID NO: 2(and therefore equivalent positions in albumins and fragments from humanserum and albumin and non-human serum albumins) may be altered in orderto modulate (increase of decrease) the binding affinity and/or half-lifee.g. plasma half-life of an albumin, fragment, fusion, conjugate,associate, nanoparticle or composition. An alteration may be asubstitution, insertion or deletion. Substitution is preferred.

A substitution or insertion may or may not comprise introduction of aconserved amino acid, i.e. conserved in relation to the amino acid atthe position of interest. Examples of conserved amino acids are shown bythe groups of FIG. 3: aliphatic, aromatic, hydrophobic, charged, polar,positive, tiny and small. At position 492 of SEQ ID NO: 2 (or equivalentposition of other albumins or variants or fragments thereof), it ispreferred that the alteration is a substitution, such as from the nativeamino acid to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y, more preferred to G, D, F, H, M or R, even more preferred to G or Dand most preferred to G. In SEQ ID NO: 2 the native amino acid atposition 492 is E, therefore a substitution to E is not preferred.

At position 550 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof), it is preferred that thealteration is a substitution, such as from the native amino acid to A,C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, more preferredto K, L, M, E or R, even more preferred to K, L or M and most preferredto K. In SEQ ID NO: 2 the native amino acid at position 550 is D,therefore a substitution to D is not preferred.

At position 573 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof), it is preferred that thealteration is a substitution, such as from the native amino acid to A,C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, more preferredto P, Y, W, H, F, T, I or V, even more preferred to P, Y or W and mostpreferred to P. In SEQ ID NO: 2 the native amino acid at position 573 isK, therefore a substitution to K is not preferred.

At position 574 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof), it is preferred that thealteration is a substitution, such as from the native amino acid to A,C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, more preferredto D, F, G, H, N, S or Y, even more preferred to H, D, F or G and mostpreferred to H. In SEQ ID NO: 2 the native amino acid at position 574 isK, therefore a substitution to K is not preferred.

At position 580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof), it is preferred that thealteration is a substitution, such as from the native amino acid to A,C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, more preferredto K or R, most preferred to K. In SEQ ID NO: 2 the native amino acid atposition 580 is Q, therefore a substitution to Q is not preferred.

A variant albumin may comprise alterations at positions corresponding topositions 492+550 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 492D+550K (e.g. SEQ ID NO: 231), or 492G+550K (e.g. SEQ ID NO:240).

A variant albumin may comprise alterations at positions corresponding topositions 492+573 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 492F+573P (e.g. SEQ ID NO: 109), 492G+573P (e.g. SEQ ID NO:110), 492H+573P (e.g. SEQ ID NO: 111) or 492R+573P (e.g. SEQ ID NO: 113)or more preferably 492D+573P (e.g. SEQ ID NO: 108). However, it ispreferred that the variant does not consist of SEQ ID NO: 2 with onlyalterations 492G+K573A, E492G+K573A, E492G+N503K+K573A,E492G+N503H+K573A, E492G+K573P, E492G+N503K+K573P or E492G+N503H+K573P.

A variant albumin may comprise alterations at positions corresponding topositions 492+574 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 492D+574H (e.g. SEQ ID NO: 232), 492G+574H (e.g. SEQ ID NO:241) or 492D+574H (e.g. SEQ ID NO: 232).

A variant albumin may comprise alterations at positions corresponding topositions 492+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof).

A variant albumin may comprise alterations at positions corresponding topositions 550+573 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise D550K+K573P (e.g. SEQ ID NO: 117).

A variant albumin may comprise alterations at positions corresponding topositions 550+574 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 550K+574H (e.g. SEQ ID NO: 130), 550M+574H (e.g. SEQ ID NO:249), 550M+574H (e.g. SEQ ID NO: 249) or 550L+574H (e.g. SEQ ID NO:245).

A variant albumin may comprise alterations at positions corresponding topositions 550+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 550K+580K (e.g. SEQ ID NO: 131), 550M+580K (e.g. SEQ ID NO:251) or 550L+580K (e.g. SEQ ID NO: 247).

A variant albumin may comprise alterations at positions corresponding topositions 573+574 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 574D+573P (e.g. SEQ ID NO: 121), 574F+573P (e.g. SEQ ID NO:122), 574G+573P (e.g. SEQ ID NO: 123), 574H+573P (e.g. SEQ ID NO: 124),574N+573P (e.g. SEQ ID NO: 125) or 5745+573P (e.g. SEQ ID NO: 126). Itis preferred that the variant does not consist of SEQ ID NO: 2 with onlyalterations K573P+K574N+A577T+A578R+S579C+Q580K+A581 D+G584A.

A variant albumin may comprise alterations at positions corresponding topositions

573+580 of SEQ ID NO: 2 (or equivalent position of other albumins orvariants or fragments thereof). Such alterations may comprise 580K+573P(e.g. SEQ ID NO: 128) or 580R+573P (e.g. SEQ ID NO: 129). However, it ispreferred that the variant does not consist of SEQ ID NO: 2 with onlyalterations K573P+A577E+A578S+Q580K+A582T.

A variant albumin may comprise alterations at positions corresponding topositions 574+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof).

A variant albumin may comprise alterations at positions corresponding topositions 492+550+573 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 492G+550K+573P (e.g. SEQ ID NO: 254) or 492D+550K+573P (e.g.SEQ ID NO: 253).

A variant albumin may comprise alterations at positions corresponding topositions 492+550+574 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise E492G+D550K+K574H (e.g. SEQ ID NO: 255).

A variant albumin may comprise alterations at positions corresponding topositions 492+550+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 492D+550K+580K (e.g. SEQ ID NO: 258) or 492G+550K+580K (e.g.SEQ ID NO: 259).

A variant albumin may comprise alterations at positions corresponding topositions 492+573+574 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 492D+573P+574H (e.g. SEQ ID NO: 233).

A variant albumin may comprise alterations at positions corresponding topositions 492+573+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 492D+573P+580K (e.g. SEQ ID NO: 234) or 492G+573P+580K (e.g.SEQ ID NO: 242).

A variant albumin may comprise alterations at positions corresponding topositions 492+574+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 492D+574H+580K (SEQ ID NO: 262) or 492G+574H+580K (e.g. SEQ IDNO: 263).

A variant albumin may comprise alterations at positions corresponding topositions 550+573+574 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 550K+574H+573P (e.g. SEQ ID NO: 131), 550L+573P+574H (e.g. SEQID NO: 246) or 550M+573P+574H (e.g. SEQ ID NO: 250).

A variant albumin may comprise alterations at positions corresponding topositions 550+573+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 550L+573P+580K (e.g. SEQ ID NO: 248) or 550M+573P+580K (e.g.SEQ ID NO: 252).

A variant albumin may comprise alterations at positions corresponding topositions 550+574+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof).

A variant albumin may comprise alterations at positions corresponding topositions 573+574+580 of SEQ ID NO: 2 (or equivalent position of otheralbumins or variants or fragments thereof). Such alterations maycomprise 574H+580K+573P (e.g. SEQ ID NO: 135).

A variant albumin may comprise alterations at positions corresponding topositions 492+550+573+574 of SEQ ID NO: 2 (or equivalent position ofother albumins or variants or fragments thereof). Such alterations maycomprise 492G+550K+573P+574H (e.g. SEQ ID NO: 257) or492D+550K+573P+574H (e.g. SEQ ID NO: 256).

A variant albumin may comprise alterations at positions corresponding topositions 492+550+573+580 of SEQ ID NO: 2 (or equivalent position ofother albumins or variants or fragments thereof). Such alterations maycomprise 492D+550K+573P+580K (e.g. SEQ ID NO: 260) or492G+550K+573P+580K (e.g. SEQ ID NO: 261).

A variant albumin may comprise alterations at positions corresponding topositions 492+550+574+580 of SEQ ID NO: 2 (or equivalent position ofother albumins or variants or fragments thereof).

A variant albumin may comprise alterations at positions corresponding topositions 492+573+574+580 of SEQ ID NO: 2 (or equivalent position ofother albumins or variants or fragments thereof). Such alterations maycomprise 492D+573P+574H+580K (e.g. SEQ ID NO: 114) or492G+573P+574H+580K (e.g. SEQ ID NO: 115), 492F+573P+574G+580K (e.g. SEQID NO: 238), 492G+573P+574G+580K (e.g. SEQ ID NO: 234),492D+573P+574G+580K (e.g. SEQ ID NO: 235), 492F+573P+574H+580R (e.g. SEQID NO: 239), 492D+573P+574H+580K (e.g. SEQ ID NO: 264),492G+573P+574H+580R (e.g. SEQ ID NO: 244), 492D+573P+574H+580R (e.g. SEQID NO: 236) or 492F+573P+574H+580K (e.g. SEQ ID NO: 237).

A variant albumin may comprise alterations at positions corresponding topositions 550+573+574+580 of SEQ ID NO: 2 (or equivalent position ofother albumins or variants or fragments thereof). Such alterations maycomprise 550K+573P+574H+580K (e.g. SEQ ID NO: 265).

A variant albumin may comprise alterations at positions corresponding topositions 492+550+573+574+580 of SEQ ID NO: 2 (or equivalent position ofother albumins or variants or fragments thereof). Such alterations maycomprise 492D+550K+573P+574H+580K (e.g. SEQ ID NO: 266) or492G+550K+573P+574H+580K (e.g. SEQ ID NO: 267). Such alterations maycomprise 492D+550K+573P+574H (e.g. SEQ ID NO: 256).

Particularly preferred variants include:

a variant albumin comprising alterations at positions corresponding topositions 492 and 580 in SEQ ID NO: 2, such as (i) E492G and Q580K, or(ii) E492D and Q580K (or equivalent positions of other albumins orvariants or fragment thereof);

a variant albumin comprising alterations at positions corresponding topositions 492 and 574 in SEQ ID NO: 2, such as (i) E492G and K574H, (ii)E492D and K574H, (iii) E492D and K574K, or (iv) E492G and K574K (orequivalent positions of other albumins or variants or fragment thereof);

a variant albumin comprising alterations at positions corresponding topositions 492 and 550 in SEQ ID NO: 2, such as (i) E492G and D550K, or(ii) E492D and D550K (or equivalent positions of other albumins orvariants or fragment thereof);

a variant albumin comprising alterations at positions corresponding topositions 550 and 573 in SEQ ID NO: 2, such as (i) D550K and K573P, (ii)D550L and K573P or (iii) D550M and

K573P (or equivalent positions of other albumins or variants or fragmentthereof); a variant albumin comprising alterations at positionscorresponding to positions 550 and 574 in SEQ ID NO: 2, such as (i)D550K and K574H, or (ii) D550L and K574H (or equivalent positions ofother albumins or variants or fragment thereof);

a variant albumin comprising alterations at positions corresponding topositions 550 and 580 in SEQ ID NO: 2, such as (i) D550M and Q580K, (ii)D550L and Q580K or (iii) D550K and Q580K (or equivalent positions ofother albumins or variants or fragment thereof);

a variant albumin with alterations (e.g. comprising alterations) atpositions corresponding to positions 580 and 573 in SEQ ID NO: 2, suchas Q580K and K573P (or equivalent positions of other albumins orvariants or fragment thereof) and preferably one or more (several) otheralterations such at a position selected from 492, 550 and 574. If thereis a K at position 580 and a P at position 573 it is preferred that thepolypeptide comprises an additional alteration at a position selectedfrom the group consisting of 492, 550, and 574.

a variant albumin with alterations (e.g. comprising alterations) atpositions corresponding to positions 492 and 573 in SEQ ID NO: 2, suchas a variant albumin comprising alterations at positions correspondingto positions 492 and 573 in SEQ ID NO: 2, such as (i) E492D and K573P or(ii) E492G and K573P or (iii) E492G and K573A (or equivalent positionsof other albumins or variants or fragment thereof), and preferably oneor more (several) other alterations such at a position selected from550, 574 and 580. If there is a G at position 492 and an A or P atposition 573 it is preferred that the polypeptide comprises anadditional alteration at a position selected from the group consistingof 550,574 and 580 (or equivalent positions of other albumins orvariants or fragment thereof);

a variant albumin with alterations (e.g. comprising alterations) atpositions corresponding to positions 573 and 574 in SEQ ID NO: 2, suchas K573P and K574H (or equivalent positions of other albumins orvariants or fragment thereof, and preferably one or more (several)alterations such as at a position selected from 492, 550 and 580. Ifthere is a P at position 573 and an N at position 574 it is preferredthat the polypeptide comprises an additional alteration at a positionselected from the group consisting of 492, 550, and 580 (or equivalentpositions of other albumins or variants or fragment thereof);

a variant albumin comprising alterations at positions corresponding topositions 574 and 580 in SEQ ID NO: 2, such as 574H and 580K (orequivalent positions of other albumins or variants or fragment thereof.If there is a N at position 574 and a K at position 580 it is preferredthat the polypeptide comprises an additional alteration at a positionselected from the group consisting of 492, 550, and 573 (or equivalentpositions of other albumins or variants or fragment thereof);

a variant albumin comprising alterations at positions corresponding topositions 492, 550 and 573 in SEQ ID NO: 2, such as E492G, D550K andK573P e.g. SEQ ID NO: 254 or such as E492D, D550K and K573P e.g. SEQ IDNO: 253;

a variant albumin comprising alterations at positions corresponding topositions 550, 573 and 580 in SEQ ID NO: 2, such as D550K, K573P andQ580K e.g. SEQ ID NO: 133;

a variant albumin comprising alterations at positions corresponding topositions 550, 573 and 580 in SEQ ID NO: 2, such as D550L, K573P andQ580K e.g. SEQ ID NO: 248 or such as D550M, K573P and Q580K e.g. SEQ IDNO:252;

a variant albumin comprising alterations at positions corresponding topositions 550, 573 and 574 in SEQ ID NO: 2, such as D550L, K573P andK574H e.g. SEQ ID NO:246;

a variant albumin comprising alterations at positions corresponding topositions 550, 573 and 574 in SEQ ID NO: 2, such as D550K, K573P andK574H e.g. SEQ ID NO: 131;

a variant albumin comprising alterations at positions corresponding topositions 492, 573 and 580 in SEQ ID NO: 2, such as E492G, K573P andQ580K e.g. SEQ ID NO: 242;

a variant albumin comprising alterations at positions corresponding topositions 492, 573 and 580 in SEQ ID NO: 2, such as E492D, K573P andQ580K e.g. SEQ ID NO: 234;

a variant albumin comprising alterations at positions corresponding topositions 492, 550, 573 and 574 in SEQ ID NO: 2, such as E492D, D550K,K573P and K574H e.g. SEQ ID NO: 256;

a variant albumin comprising alterations at positions corresponding topositions 492, 550, 573 and 574 in SEQ ID NO: 2, such as E492G, D550K,K573P and K574H e.g. SEQ ID NO: 257;

a variant albumin comprising alterations at positions corresponding topositions 573, 574 and 580 in SEQ ID NO: 2, such as K573P, K574H andQ580K e.g. SEQ ID NO: 135;

a variant albumin comprising alterations at positions corresponding topositions 492, 573, 574 and 580 in SEQ ID NO: 2, such as E492D, K573P,K574H and Q580K (or equivalent positions of other albumins or variantsor fragment thereof), e.g. SEQ ID NO: 114;

a variant albumin comprising alterations at positions corresponding topositions 492, 573, 574 and 580 in SEQ ID NO: 2, such as E492G, K573P,K574H and Q580K (or equivalent positions of other albumins or variantsor fragment thereof), e.g. SEQ ID NO: 115.

It is preferred that the alteration at position 492 is conservedrelative to D or E. It is preferred that the alteration at position 574is conserved relative to H. It is preferred that the alteration atposition 580 is conserved relative to K.

Advantageously, the polypeptide retains substantially the same tertiarystructure (or, for a fragment, the relevant part of the structure) as areference or parent albumin such as HSA. The skilled person understandthe term ‘substantially the same tertiary structure’ bearing in mindthat some degree of variation in tertiary structure is expected as allproteins have some degree of structural flexibility. This appliesparticularly to polypeptides having a higher binding affinity to FcRnthan the parent or reference albumin (e.g. HSA) has to FcRn.

One or more (several) of the His residues may or may not be maintainedrelative to the parent albumin. For example, with reference to SEQ IDNO: 2, one or more (several) of the following His residues may bemaintained: 3, 9, 39, 67, 105, 128, 146, 242, 247, 288, 338, 367, 440,464, 510, 535. One or more (several), preferably all, of the Hisresidues in domain I are maintained (i.e. 3, 9, 39, 67, 105, 128, 146.).One or more (several), preferably all, of the His residues in domain IIare maintained (i.e. 242, 247, 288, 338, 367). One or more (several),preferably all, of the His residues in domain III are maintained (i.e.440, 464, 510, 535). One or more (several) or all three of His 464, 510,535 may be maintained.

It is preferred that at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16 or 17 of the disulphide bonds of the albumin are maintainedin the polypeptide. For a polypeptide derived from a full lengthalbumin, it is preferred that all disulphide bonds usually present inthat albumin are maintained. For a polypeptide derived from a fragmentof albumin, it is preferred that all disulphide bonds usually present inthat fragment are maintained. It is preferred that Cys34 (or equivalentin non-human albumins) is maintained.

For all aspects of the invention fusion partner polypeptides and/orconjugates may comprise one or more (several) of: 4-1BB ligand, 5-helix,A human C—C chemokine, A human L105 chemokine, A human L105 chemokinedesignated huL105_(—)3., A monokine induced by gamma-interferon (MIG), Apartial CXCR4B protein, A platelet basic protein (PBP), a1-antitrypsin,ACRP-30 Homologue; Complement Component Clq C, Adenoid-expressedchemokine (ADEC), aFGF; FGF-1, AGF, AGF Protein, albumin, an etoposide,angiostatin, Anthrax vaccine, Antibodies specific for collapsin,antistasin, Anti-TGF beta family antibodies, antithrombin III, APM-1;ACRP-30; Famoxin, apo-lipoprotein species, Arylsulfatase B, b57 Protein,BCMA, Beta-thromboglobulin protein (beta-TG), bFGF; FGF2, Bloodcoagulation factors, BMP Processing Enzyme Furin, BMP-10, BMP-12,BMP-15, BMP-17, BMP-18, BMP-2B, BMP-4, BMP-5, BMP-6, BMP-9, BoneMorphogenic Protein-2, calcitonin, Calpain-10a, Calpain-10b,Calpain-10c, Cancer Vaccine, Carboxypeptidase, C—C chemokine, MCP2, CCR5variant, CCR7, CCR7, CD11a Mab, CD137; 4-1BB Receptor Protein, CD20 Mab,CD27, CD27L, CD30, CD30 ligand, CD33 immunotoxin, CD40, CD40L, CD52 Mab,Cerebus Protein, Chemokine Eotaxin., Chemokine hIL-8, Chemokine hMCP1,Chemokine hMCP1a, Chemokine hMCP1b, Chemokine hMCP2, Chemokine hMCP3,Chemokine hSDF1b, Chemokine MCP-4, chemokine TECK and TECK variant,Chemokine-like protein IL-8M1 Full-Length and Mature, Chemokine-likeprotein IL-8M10 Full-Length and Mature, Chemokine-like protein IL-8M3,Chemokine-like protein IL-8M8 Full-Length and Mature, Chemokine-likeprotein IL-8M9 Full-Length and Mature, Chemokine-like protein PF4-414Full-Length and Mature, Chemokine-like protein PF4-426 Full-Length andMature, Chemokine-like protein PF4-M2 Full-Length and Mature, Choleravaccine, Chondromodulin-like protein, c-kit ligand; SCF; Mast cellgrowth factor; MGF; Fibrosarcoma-derived stem cell factor, CNTF andfragment thereof (such as CNTFAx15′(Axokine™)), coagulation factors inboth pre and active forms, collagens, Complement C5 Mab, Connectivetissue activating protein-Ill, CTAA16.88 Mab, CTAP-III, CTLA4-Ig,CTLA-8, CXC3, CXC3, CXCR3; CXC chemokine receptor 3, cyanovirin-N,Darbepoetin, designated exodus, designated huL105_(—)7., DIL-40, DNase,EDAR, EGF Receptor Mab, ENA-78, Endostatin, Eotaxin, Epithelialneutrophil activating protein-78, EPO receptor; EPOR, erythropoietin(EPO) and EPO mimics, Eutropin, Exodus protein, Factor IX, Factor VII,Factor VIII, Factor X and Factor XIII, FAS Ligand Inhibitory Protein(DcR3), FasL, FasL, FasL, FGF, FGF-12; Fibroblast growth factorhomologous factor-1, FGF-15, FGF-16, FGF-18, FGF-3; INT-2, FGF-4;gelonin, HST-1; HBGF-4, FGF-5, FGF-6; Heparin binding secretedtransforming factor-2, FGF-8, FGF-9; Glia activating factor, fibrinogen,flt-1, flt-3 ligand, Follicle stimulating hormone Alpha subunit,Follicle stimulating hormone Beta subunit, Follitropin, Fractalkine,fragment. myofibrillar protein Troponin I, FSH, Galactosidase,Galectin-4, G-CSF, GDF-1, Gene therapy, Glioma-derived growth factor,glucagon, glucagon-like peptides, Glucocerebrosidase, glucose oxidase,Glucosidase, Glycodelin-A; Progesterone-associated endometrial protein,GM-CSF, gonadotropin, Granulocyte chemotactic protein-2 (GCP-2),Granulocyte-macrophage colony stimulating factor, growth hormone, Growthrelated oncogene-alpha (GRO-alpha), Growth related oncogene-beta(GRO-beta), Growth related oncogene-gamma (GRO-gamma), hAPO-4; TROY,hCG, Hepatitus B surface Antigen, Hepatitus B Vaccine, HER2Receptor Mab,hirudin, HIV gp120, HIV gp41, HIV Inhibitor Peptide, HIV InhibitorPeptide, HIV Inhibitor Peptide, HIV protease inhibiting peptides, HIV-1protease inhibitors, HPV vaccine, Human 6CKine protein, Human Act-2protein, Human adipogenesis inhibitory factor, human B cell stimulatingfactor-2 receptor, Human beta-chemokine H1305 (MCP-2), Human C—Cchemokine DGWCC, Human CC chemokine ELC protein, Human CC type chemokineinterleukin C, Human CCC3 protein, Human CCF18 chemokine, Human CC-typechemokine protein designated SLC (secondary lymphoid chemokine), Humanchemokine beta-8 short forms, Human chemokine C10, Human chemokine CC-2,Human chemokine CC-3, Human chemokine CCR-2, Human chemokine Ckbeta-7,Human chemokine ENA-78, Human chemokine eotaxin, Human chemokine GROalpha, Human chemokine GROalpha, Human chemokine GRObeta, Humanchemokine HCC-1, Human chemokine HCC-1, Human chemokine 1-309, Humanchemokine IP-10, Human chemokine L105_(—)3, Human chemokine L105_(—)7,Human chemokine MIG, Human chemokine MIG-beta protein, Human chemokineMIP-1alpha, Human chemokine MIP1beta, Human chemokine MIP-3alpha, Humanchemokine MIP-3beta, Human chemokine PF4, Human chemokine protein 331D5,Human chemokine protein 61164, Human chemokine receptor CXCR3, Humanchemokine SDF1alpha, Human chemokine SDF1beta, Human chemokine ZSIG-35,Human Chr19Kine protein, Human CKbeta-9, Human CKbeta-9, Human CX3C 111amino acid chemokine, Human DNAX interleukin-40, Human DVic-1 C—Cchemokine, Human EDIRF I protein sequence, Human EDIRF II proteinsequence, Human eosinocyte CC type chemokine eotaxin, Humaneosinophil-expressed chemokine (EEC), Human fast twitch skeletal muscletroponin C, Human fast twitch skeletal muscle troponin I, Human fasttwitch skeletal muscle Troponin subunit C, Human fast twitch skeletalmuscle Troponin subunit I Protein, Human fast twitch skeletal muscleTroponin subunit T, Human fast twitch skeletal muscle troponin T, Humanfoetal spleen expressed chemokine, FSEC, Human GM-CSF receptor, Humangro-alpha chemokine, Human gro-beta chemokine, Human gro-gammachemokine, Human IL-16 protein, Human IL-1RD10 protein sequence, HumanIL-1RD9, Human IL-5 receptor alpha chain, Human IL-6 receptor, HumanIL-8 receptor protein hIL8RA, Human IL-8 receptor protein hIL8RB, HumanIL-9 receptor protein, Human IL-9 receptor protein variant #3, HumanIL-9 receptor protein variant fragment, Human IL-9 receptor proteinvariant fragment#3, Human interleukin 1 delta, Human Interleukin 10,Human Interleukin 10, Human interleukin 18, Human interleukin 18derivatives, Human interleukin-1 beta precursor, Human interleukin-1beta precursor., Human interleukin-1 receptor accessory protein, Humaninterleukin-1 receptor antagonist beta, Human interleukin-1 type-3receptor, Human Interleukin-10 (precursor), Human Interleukin-10(precursor), Human interleukin-11 receptor, Human interleukin-12 40 kDsubunit, Human interleukin-12 beta-1 receptor, Human interleukin-12beta-2 receptor, Human Interleukin-12 p35 protein, Human Interleukin-12p40 protein, Human interleukin-12 receptor, Human interleukin-13 alphareceptor, Human interleukin-13 beta receptor, Human interleukin-15,Human interleukin-15 receptor from clone P1, Human interleukin-17receptor, Human interleukin-18 protein (IL-18), Human interleukin-3,human interleukin-3 receptor, Human interleukin-3 variant, Humaninterleukin-4 receptor, Human interleukin-5, Human interleukin-6, Humaninterleukin-7, Human interleukin-7., Human interleukin-8 (IL-8), Humanintracellular IL-1 receptor antagonist, Human IP-10 and HIV-1 gp120hypervariable region fusion protein, Human IP-10 and human Muc-1 coreepitope (VNT) fusion protein, human liver and activation regulatedchemokine (LARC), Human Lkn-1 Full-Length and Mature protein, Humanmammary associated chemokine (MACK) protein Full-Length and Mature,Human mature chemokine Ckbeta-7, Human mature gro-alpha, Human maturegro-gamma polypeptide used to treat sepsis, Human MCP-3 and human Muc-1core epitope (VNT) fusion protein, Human MI10 protein, Human MI1Aprotein, Human monocyte chemoattractant factor hMCP-1, Human monocytechemoattractant factor hMCP-3, Human monocyte chemotactic proprotein(MCPP) sequence, Human neurotactin chemokine like domain, Human non-ELRCXC chemokine H174, Human non-ELR CXC chemokine IP10, Human non-ELR CXCchemokine Mig, Human PAI-1 mutants, Human protein with IL-16 activity,Human protein with IL-16 activity, Human secondary lymphoid chemokine(SLC), Human SISD protein, Human STCP-1, Human stromal cell-derivedchemokine, SDF-1, Human T cell mixed lymphocyte reaction expressedchemokine (TMEC), Human thymus and activation regulated cytokine (TARC),Human thymus expressed, Human TNF-alpha, Human TNF-alpha, Human TNF-beta(LT-alpha), Human type CC chemokine eotaxin 3 protein sequence, Humantype II interleukin-1 receptor, Human wild-type interleukin-4 (hIL-4)protein, Human ZCHEMO-8 protein, Humanized Anti-VEGF Antibodies, andfragments thereof, Humanized Anti-VEGF Antibodies, and fragmentsthereof, Hyaluronidase, ICE 10 kD subunit., ICE 20 kD subunit., ICE 22kD subunit., Iduronate-2-sulfatase, Iduronidase, IL-1 alpha, IL-1 beta,IL-1 inhibitor (IL-1i)., IL-1 mature, IL-10 receptor, IL-11, IL-11,IL-12 p40 subunit., IL-13, IL-14, IL-15, IL-15 receptor, IL-17, IL-17receptor, IL-17 receptor, IL-17 receptor, IL-19, IL-1i fragments,IL1-receptor antagonist, IL-21 (TIF), IL-3 containing fusion protein.,IL-3 mutant proteins, IL-3 variants, IL-3 variants, IL-4, IL-4 mutein,IL-4 mutein Y124G, IL-4 mutein Y124X, IL-4 muteins, 11-5 receptor, IL-6,11-6 receptor, IL-7 receptor clone, IL-8 receptor, IL-9 mature proteinvariant (Met117 version), immunoglobulins or immunoglobulin-basedmolecules or fragment of either (e.g. a Small ModularImmunoPharmaceutical™ (“SMIP”) or dAb, Fab′ fragments, F(ab′)2, scAb,scFv or scFv fragment), including but not limited to plasminogen,Influenza Vaccine, Inhibin alpha, Inhibin beta, insulin, insulin-likegrowth factor, Integrin Mab, inter-alpha trypsin inhibitor, inter-alphatrypsin inhibitor, Interferon gamma-inducible protein (IP-10),interferons (such as interferon alpha species and sub-species,interferon beta species and sub-species, interferon gamma species andsub-species), interferons (such as interferon alpha species andsub-species, interferon beta species and sub-species, interferon gammaspecies and sub-species), Interleukin 6, Interleukin 8 (IL-8) receptor,Interleukin 8 receptor B, Interleukin-1alpha, Interleukin-2 receptorassociated protein p43, interleukin-3, interleukin-4 muteins,Interleukin-8 (IL-8) protein., interleukin-9, Interleukin-9 (IL-9)mature protein (Thr117 version), interleukins (such as M0, IL11 andIL2), interleukins (such as IL10, IL11 and IL2), Japanese encephalitisvaccine, Kalikrein Inhibitor, Keratinocyte growth factor, Kunitz domainprotein (such as aprotinin, amyloid precursor protein and thosedescribed in WO 03/066824, with or without albumin fusions), Kunitzdomain protein (such as aprotinin, amyloid precursor protein and thosedescribed in WO 03/066824, with or without albumin fusions), LACI,lactoferrin, Latent TGF-beta binding protein II, leptin, Liver expressedchemokine-1 (LVEC-1), Liver expressed chemokine-2 (LVEC-2), LT-alpha,LT-beta, Luteinization Hormone, Lyme Vaccine, Lymphotactin, Macrophagederived chemokine analogue MDC (n+1), Macrophage derived chemokineanalogue MDC-eyfy, Macrophage derived chemokine analogue MDC-yl,Macrophage derived chemokine, MDC, Macrophage-derived chemokine (MDC),Maspin; Protease Inhibitor 5, MCP-1 receptor, MCP-1a, MCP-1b, MCP-3,MCP-4 receptor, M-CSF, Melanoma inhibiting protein, Membrane-boundproteins, Met117 human interleukin 9, MIP-3 alpha, MIP-3 beta,MIP-Gamma, MIRAP, Modified Rantes, monoclonal antibody, MP52, MutantInterleukin 6 S176R, myofibrillar contractile protein Troponin I,Natriuretic Peptide, Nerve Growth Factor-beta, Nerve GrowthFactor-beta2, Neuropilin-1, Neuropilin-2, Neurotactin, Neurotrophin-3,Neurotrophin-4, Neurotrophin-4-a, Neurotrophin-4-b, Neurotrophin-4-c,Neurotrophin-4-d, Neutrophil activating peptide-2 (NAP-2), NOGO-66Receptor, NOGO-A, NOGO-B, NOGO-C, Novel beta-chemokine designated PTEC,N-terminal modified chemokine GroHEK/hSDF-1alpha, N-terminal modifiedchemokine GroHEK/hSDF-1beta., N-terminal modified chemokine met-hSDF-1alpha, N-terminal modified chemokine met-hSDF-1 beta, OPGL, OsteogenicProtein-1; OP-1; BMP-7, Osteogenic Protein-2, OX40; ACT-4, OX40L,Oxytocin (Neurophysin I), parathyroid hormone, Patched, Patched-2,PDGF-D, Pertussis toxoid, Pituitary expressed chemokine (PGEC),Placental Growth Factor, Placental Growth Factor-2, PlasminogenActivator Inhibitor-1; PAI-1, Plasminogen Activator Inhibitor-2; PAI-2,Plasminogen Activator Inhibitor-2; PAI-2, Platelet derived growthfactor, Platelet derived growth factor Bv-sis, Platelet derived growthfactor precursor A, Platelet derived growth factor precursor B, PlateletMab, platelet-derived endothelial cell growth factor (PD-ECGF),Platelet-Derived Growth Factor A chain, Platelet-Derived Growth Factor Bchain, polypeptide used to treat sepsis, Preproapolipoprotein “milano”variant, Preproapolipoprotein “paris” variant, pre-thrombin, Primate CCchemokine “ILINCK”, Primate CXC chemokine “IBICK”, proinsulin,Prolactin, Prolactin2, prosaptide, Protease inhibitor peptides, ProteinC, Protein S, pro-thrombin, prourokinase, RANTES, RANTES 8-68, RANTES9-68, RANTES peptide, RANTES receptor, Recombinant interleukin-16,Resistin, restrictocin, Retroviral protease inhibitors, ricin, RotavirusVaccine, RSV Mab, saporin, sarcin, Secreted and Transmembranepolypeptides, Secreted and Transmembrane polypeptides, serumcholinesterase, serum protein (such as a blood clotting factor), SolubleBMP Receptor Kinase Protein-3, Soluble VEGF Receptor, Stem CellInhibitory Factor, Straphylococcus Vaccine, Stromal Derived Factor-1alpha, Stromal Derived Factor-1 beta, Substance P (tachykinin), T1249peptide, T20 peptide, T4 Endonuclease, TACI, Tarc, TGF-beta 1, TGF-beta2, Thr117 human interleukin 9, thrombin, thrombopoietin, Thrombopoietinderivative1, Thrombopoietin derivative2, Thrombopoietin derivative3,Thrombopoietin derivative4, Thrombopoietin derivative5, Thrombopoietinderivative6, Thrombopoietin derivative7, Thymus expressed chemokine(TECK), Thyroid stimulating Hormone, tick anticoagulant peptide, Tim-1protein, TNF-alpha precursor, TNF-R, TNF-R11; TNF p75 Receptor; DeathReceptor, tPA, transferrin, transforming growth factor beta, Troponinpeptides, Truncated monocyte chemotactic protein 2 (6-76), Truncatedmonocyte chemotactic protein 2 (6-76), Truncated RANTES protein (3-68),tumour necrosis factor, Urate Oxidase, urokinase, Vasopressin(Neurophysin II), VEGF R-3; flt-4, VEGF Receptor; KDR; flk-1, VEGF-110,VEGF-121, VEGF-138, VEGF-145, VEGF-162, VEGF-165, VEGF-182, VEGF-189,VEGF-206, VEGF-D, VEGF-E; VEGF-X, von Willebrand's factor, Wild typemonocyte chemotactic protein 2, Wild type monocyte chemotactic protein2, ZTGF-beta 9, alternative antibody scaffolds e.g. anticalin(s),adnectin(s), fibrinogen fragment(s), nanobodies such as camelidnanobodies, infestin, and/or any of the molecules mentioned inWO01/79271 (particularly page 9 and/or Table 1), WO 2003/59934(particularly Table 1), WO03/060071 (particularly Table 1) orWO01/079480 (particularly Table 1) (each incorporated herein byreference in their entirety).

Furthermore, conjugates may comprise one or more (several) ofchemotherapy drugs such as: 13-cis-Retinoic Acid, 2-CdA,2-Chlorodeoxyadenosine, 5-Azacitidine, 5-Fluorouracil, 5-FU,6-Mercaptopurine, 6-MP, 6-TG, 6-Thioguanine, A, Abraxane, Accutane®,Actinomycin-D, Adriamycin®, Adrucil®, Agrylin®, Ala-Cort®, Aldesleukin,Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ®, Alkeran®,All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin,Amifostine, Aminoglutethimide, Anagrelide, Anandron®, Anastrozole,Arabinosylcytosine, Ara-C, Aranesp®, Aredia®, Arimidex®, Aromasin®,Arranon®, Arsenic Trioxide, Asparaginase, ATRA, Avastin®, Azacitidine,BCG, BCNU, Bevacizumab, Bexarotene, BEXXAR®, Bicalutamide, BiCNU,Blenoxane®, Bleomycin, Bortezomib, Busulfan, Busulfex®, C225, CalciumLeucovorin, Campath®, Camptosar®, Camptothecin-11, Capecitabine, Carac™,Carboplatin, Carmustine, Carmustine Wafer, Casodex®, CC-5013, CCNU,CDDP, CeeNU, Cerubidine®, Cetuximab, Chlorambucil, Cisplatin, CitrovorumFactor, Cladribine, Cortisone, Cosmegen®, CPT-11, Cyclophosphamide,Cytadren®, Cytarabine, Cytarabine Liposomal, Cytosar-U®, Cytoxan®,Dacarbazine, Dacogen, Dactinomycin, Darbepoetin Alfa, Dasatinib,Daunomycin, Daunorubicin, Daunorubicin Hydrochloride, DaunorubicinLiposomal, DaunoXome®, Decadron, Decitabine, Delta-Cortef®, Deltasone®,Denileukin diftitox, DepoCyt™, Dexamethasone, Dexamethasone acetate,Dexamethasone Sodium Phosphate, Dexasone, Dexrazoxane, DHAD, DIC,Diodex, Docetaxel, Doxil®, Doxorubicin, Doxorubicin liposomal, Droxia™,DTIC, DTIC-Dome®, Duralone®, Efudex®, Eligard™, Ellence™, Eloxatin™,Elspar®, Emcyt®, Epirubicin, Epoetin alfa, Erbitux™, Erlotinib, ErwiniaL-asparaginase, Estramustine, Ethyol, Etopophos®, Etoposide, EtoposidePhosphate, Eulexin®, Evista®, Exemestane, Fareston®, Faslodex®, Femara®,Filgrastim, Floxuridine, Fludara®, Fludarabine, Fluoroplex®,Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, FolinicAcid, FUDR®, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumabozogamicin, Gemzar®, Gleevec™, Gliadel® Wafer, GM-CSF, Goserelin,Granulocyte—Colony Stimulating Factor, Granulocyte Macrophage ColonyStimulating Factor, Halotestin®, Herceptin®, Hexadrol, Hexylen®,Hexamethylmelamine, HMM, Hycamtin®, Hydrea®, Hydrocort Acetate®,Hydrocortisone, Hydrocortisone Sodium Phosphate, Hydrocortisone SodiumSuccinate, Hydrocortone Phosphate, Hydroxyurea, Ibritumomab, IbritumomabTiuxetan, Idamycin®, Idarubicin, Ifex®, IFN-alpha, Ifosfamide, IL-11,IL-2, Imatinib mesylate, Imidazole Carboxamide, Interferon alfa,Interferon Alfa-2b (PEG Conjugate), Interleukin-2, Interleukin-11,Intron A® (interferon alfa-2b), Iressa®, Irinotecan, Isotretinoin,Kidrolase®, Lanacort®, Lapatinib, L-asparaginase, LCR, Lenalidomide,Letrozole, Leucovorin, Leukeran, Leukine™, Leuprolide, Leurocristine,Leustatin™, Liposomal Ara-C, Liquid Pred®, Lomustine, L-PAM,L-Sarcolysin, Lupron®, Lupron Depot®, M, Matulane®, Maxidex,Mechlorethamine, Mechlorethamine Hydrochloride, Medralone®, Medrol®,Megace®, Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna,Mesnex™, Methotrexate, Methotrexate Sodium, Methylprednisolone,Meticorten®, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol®, MTC,MTX, Mustargen®, Mustine, Mutamycin®, Myleran®, Mylocel™, Mylotarg®,Navelbine®, Nelarabine, Neosar®, Neulasta™, Neumega®, Neupogen®,Nexavar®, Nilandron®, Nilutamide, Nipent®, Nitrogen Mustard, Novaldex®,Novantrone®, Octreotide, Octreotide acetate, Oncospar®, Oncovin®,Ontak®, Onxal™, Oprevelkin, Orapred®, Orasone®, Oxaliplatin, a taxol ortaxol derivative e.g. Paclitaxel or Paclitaxel Protein-bound,Pamidronate, Panitumumab, Panretin®, Paraplatin®, Pediapred®, PEGInterferon, Pegaspargase, Pegfilgrastim, PEG-INTRON™,PEG-L-asparaginase, PEMETREXED, Pentostatin, Phenylalanine Mustard,Platinol®, Platinol-AQ®, Prednisolone, Prednisone, Prelone®,Procarbazine, PROCRIT®, Proleukin®, Prolifeprospan 20 with CarmustineImplant, Purinethol®, R, Raloxifene, Revlimid®, Rheumatrex®, Rituxan®,Rituximab, Roferon-A® (Interferon Alfa-2a), Rubex®, Rubidomycinhydrochloride, Sandostatin®, Sandostatin LAR®, Sargramostim,Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCEL™, STI-571, Streptozocin,SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®, Targretin®, Taxol®,Taxotere®, Temodar®, Temozolomide, Teniposide, TESPA, Thalidomide,Thalomid®, TheraCys®, Thioguanine, Thioguanine Tabloid®,Thiophosphoamide, Thioplex®, Thiotepa, TICE®, Toposar®, Topotecan,Toremifene, Tositumomab, Trastuzumab, Tretinoin, Trexall™, Trisenox®,TSPA, TYKERB®, VCR, Vectibix™, Velban®, Velcade®, VePesid®, Vesanoid®,Viadur™, Vidaza®, Vinblastine, Vinblastine Sulfate, Vincasar Pfs®,Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VM-26, Vorinostat,VP-16, Vumon®, Xeloda®, Zanosar®, Zevalin™ Zinecard®, Zoladex®,Zoledronic acid, Zolinza, Zometa®; radiopharmaceuticals such as:Carbon-11, Carbon-14, Chromium-51, Cobalt-57, Cobalt-58, Erbium-169,Fluorine-18, Gallium-67, Gold-198, Indium-111, Indium-113m, Iodine-123,Iodine-125, Iodine-131, Iron-59, Krypton-81m, Nitrogen-13, Oxygen-15,Phosphorous-32, Rhenium-186, Rubidium-82, Samarium-153, Selenium-75,Strontium-89, Technetium-99m, Thallium-201, Tritium, Xenon-127,Xenon-133, Yttrium-90; imaging agents such as Gadolinium, magnetite,manganese, technetium, 1125, 1131, P32, T1201, Iopamidol, PET-FDG.

Further fusion partners, conjugation partners and/or molecules forinclusion in a nanoparticle, associate or composition according to theinvention include: acromegaly drugs e.g. somatuline, lanreotide,octreotide, Sandostatin; antithrombotics e.g. bivalirudin, Angiomax,dalteparin, Fragmin, enoxaparin, Lovenox, Drotrecogin alfa (e.g.Activated), Xigris, heparin; assisted reproductive therapy compoundse.g. choriogonadotropin, Ovidrel, follitropin, alpha/beta; enzymes e.g.hyaluronidase, Hylenex; diabetes drugs e.g. exenatide, Byetta, glucagon,insulin, liraglutide, albiglutide, GLP-1 agonists, exendin or an exendinanalog; compounds useful in diagnosis e.g. protirelin, Thyrel TRHThypinone, secretin (e.g. synthetic human), Chirhostim, thyrotropin(e.g. alpha), Thyrogen' erythropoiesis drugs e.g. Darbepoetin alfa,Aranesp, Epoetin alfa, Epogen, Eprex, drugs for the treatment of geneticdefects e.g. pegademase, drugs for the treatment of growth failure e.g.Adagen, mecasermin, rinfabate, drugs for the treatment of cysticfibrosis e.g. Dornase alfa, Pulmozyme, drugs for the treatment ofmetaoblic disorders e.g. Agalsidase beta, Fabrazyme, alglucosidasealpha, Myozyme, Laronidase, Aldurazyme, drugs for the treatment ofgenital wart intralesional e.g. Interferon alfa-n3, Alferon N, drugs forthe treatment of granulomatous disease e.g. Interferon gamma-1b,Actimmune; drugs for the treatment of growth failure e.g. pegvisomant,Somavert, somatropin, Genotropin, Nutropin, Humatrope, Serostim,Protropin; drugs for the treatment of heart failure e.g. nesiritide,Natrecor; drugs for the treatment of hemophilia e.g. a coagulationfactor e.g. Factor VIII, Helixate FS, Kogenate FS, Factor IX, BeneFIX,Factor Vila, Novoseven, desmopressin, Stimate, DDAVP; hemopoetic drugse.g. Filgrastim (G-CSF), Neupogen, Oprelvekin, Neumega, Pegfilgrastim,Neulasta, Sargramostim, Leukine; drugs for the treatment of hepatitis Ce.g. Interferon alfa-2a, Roferon A, Interferon alfa-2b, Intron A,Interferon alfacon-1, Infergen, Peginterferon alfa-2a, Pegasys,Peginterferon alfa-2b, PEG-Intron; drugs for the treatment of HIV e.g.enfuvirtide, Fuzeon; Fabs e.g. Fab (antithrombin), Abciximab, ReoPro;monoclonal antibodies e.g. Daclizumab, Zenapax; antiviral monoclonalantibodies e.g. Palivizumab, Synagis; monoclonal antibodies for thetreatment of asthma e.g. Omalizumab, Xolair; monoclonal antibodies foruse in diagnostic imaging e.g. Arcitumomab, CEA-Scan, CapromabPendetide, ProstaScint, Satumomab Pendetide, OncoScint CR/OV, Fabs foruse in diagnostic imaging e.g. Nofetumomab, Verluma; iimmuno-supressantmonoclonal antibodies e.g. Basiliximab, Simulect, Muromonab-CD3,Orthoclone OKT3; monoclonal antibodies for the treatment of malignancye.g. Alemtuzumab, Campath, Ibritumomab tiuxetan, Zevalin, Rituximab,Rituxan, Trastuzumab, Herceptin; monoclonal antibodies for the treatmentof rheumatoid arthritis (RA) e.g. Adalimumab, Humira, Infliximab,Remicade; monoclonal antibodies for use as a radio-immuno-therapeutice.g. Tositumomab and Iodine I¹³¹, Tositumomab, Bexxar; drugs for thetreatment of macular degeneration e.g. pegaptanib, Macugen; drugs forthe treatment of malignancy e.g. Aldesleukin, Proleukin, Interleukin-2,Asparaginase, Elspar, Rasburicase, Elitek, Denileukin diftitox, Ontak,Pegaspargase, Oncaspar, goserelin, leuprolide; drugs for the treatmentof multiple sclerosis (MS) e.g. Glatiramer acetate (e.g. copolymer-1),Copaxone, Interferon beta-1a, Avonex, Interferon beta-1a, Rebif,Interferon beta-1b, Betaseron; drugs for the treatment of mucositis e.g.palifermin, Kepivance; drug for the treatment of dystonia e.g.,neurotoxin, Botulinum Toxin Type A, BOTOX, BOTOX Cosmetic, BotulinumToxin Type B, MYOBLOC; drugs for the treatment of osteoporosis e.g.teriparatide,Forteo; drugs for the treatment of psoriasis e.g.Alefacept, Amevive; drugs for the treatment of RA e.g. abatacept,Orencia, Anakinra, Kineret, Etanercept, Enbrel; thrombolytics e.g.Alteplase, Activase, rtPA, Anistreplase, Eminase, Reteplase, Retavase,Streptokinase, Streptase, Tenecteplase, TNKase, Urokinase, Abbokinase,Kinlytic; drugs for the treatment of osteoporosis e.g. calcitonin (e.g.salmon), Miacalcin, Fortical, drugs for the treatment of skin ulcerse.g. Becaplermin, Regranex, Collagenase, Santyl.

Such polypeptides and chemical compounds may be referred to asdiagnostic moieties, therapeutic moieties, prophylactic moieties orbeneficial moieties.

Preferably the fusion partner and/or conjugation partner is not analbumin, variant or fragment thereof.

One or more (several) therapeutic or prophylactic polypeptides may befused to the N-terminus, the C-terminus of albumin, inserted into a loopin the albumin structure or any combination thereof. It may or it maynot comprise linker sequences separating the various components of thefusion polypeptide.

Teachings relating to fusions of albumin or a fragment thereof are knownin the art and the skilled person will appreciate that such teachingscan also be applied to the invention. WO 2001/79271A and WO 2003/59934(incorporated herein by reference) also contain examples of therapeuticand prophylactic polypeptides that may be fused to albumin or fragmentsthereof, and these examples apply also to the invention.

The invention is further defined in the following embodiments:

1. A polypeptide which is a variant of albumin, fragment thereof orfusion polypeptide comprising said variant albumin or a fragment thereofhaving an altered binding affinity to FcRn compared with the bindingaffinity of a parent albumin, reference albumin, fragment thereof orfusion polypeptide comprising said parent albumin, reference albumin orfragment or fusion thereof to FcRn, wherein the polypeptide comprisesalterations at two or more positions selected from positionscorresponding to positions 492, 550, 573, 574 and 580 in SEQ ID NO: 2.2. The polypeptide according to embodiment 1 wherein the polypeptidecomprises alterations at two or more positions selected from positionscorresponding to positions (a) 492 and 580; (b) 492 and 574; (c) 492 and550; (d) 550 and 573; (e) 550 and 574; (f) 550 and 580 in SEQ ID NO: 2.3. The polypeptide according to embodiment 1 or 2 comprising alterationsat positions corresponding to positions 492 and 580 of SEQ ID NO: 1,further comprising one or more (several) alterations at positionscorresponding to positions selected from the group consisting of 550,573 and 574 of SEQ ID NO: 2.4. The polypeptide according to embodiment 1, 2 or 3 comprisingalterations at positions corresponding to positions 492 and 574 of SEQID NO: 1, further comprising one or more (several) alterations atpositions corresponding to positions selected from the group consisting550, 573 and 580 of SEQ ID NO:5. The polypeptide according to any preceding embodiment comprisingalterations at positions corresponding to positions 492 and 550 of SEQID NO: 1, further comprising one or more (several) alterations atpositions corresponding to positions selected from the group consisting573, 574 and 580 of SEQ ID NO: 2.6. The polypeptide according to any preceding embodiment comprisingalterations at positions corresponding to positions 550 and 573 of SEQID NO: 1, further comprising one or more (several) alterations atpositions corresponding to positions selected from the group consisting492, 574, and 580 of SEQ ID NO: 2.7. The polypeptide according to any preceding embodiment comprisingalterations at positions corresponding to positions 550 and 574 of SEQID NO: 1, further comprising one or more (several) alterations atpositions corresponding to positions selected from the group consisting492, 573 and 580 of SEQ ID NO: 2.8. The polypeptide according to any preceding embodiment comprisingalterations at positions corresponding to positions 550 and 580 of SEQID NO: 1, further comprising an alteration at a position correspondingto position 492, 573 and 574 of SEQ ID NO: 2.9. The polypeptide according to any preceding embodiment in which:(a) at a position corresponding to position 492 of SEQ ID NO: 2 there isan alteration to generate an amino acid from the group consisting of A,C, D, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, preferably D and at aposition corresponding to position 573 of SEQ ID NO: 2 there is analteration to generate an amino acid from the group consisting of C, D,E, F, G, H, I, L, M, N, Q, R, S, T, V, W, Y, preferably Y, W or H, or(b) at a position corresponding to position 492 of SEQ ID NO: 2 there isan alteration to generate G and at a position corresponding to position573 there is an alteration to generate A or P and the polypeptidecomprises an additional alteration at a position selected from the groupconsisting of 550, 574 and 580 (or equivalent positions of otheralbumins or variants or fragment thereof).10. The polypeptide according to any preceding embodiment in which:(a) at a position corresponding to position 573 of SEQ ID NO: 2 there isan alteration to generate an amino acid from the group consisting of A,C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, Y, preferably Y, W or Hand at a position corresponding to position 574 of SEQ ID NO: 2 there isan alteration to generate an amino acid from the group consisting of A,C, D, E, F, G, H, I, L, M, P, Q, R, S, T, V, W, Y, H, D, F, G, N, S orY, more preferably H, D, F or G, most preferably H, or(b) at a position corresponding to position 573 of SEQ ID NO: 2 there isa P and at a position corresponding to position 574 of SEQ ID NO: 2there is an N and the polypeptide comprises an additional alteration ata position selected from the group consisting of 492, 550, and 580 (orequivalent positions of other albumins or variants or fragment thereof);11. The polypeptide according to any preceding embodiment in which:(a) at a position corresponding to position 573 of SEQ ID NO: 2 there isan alteration to generate an amino acid from the group consisting of A,C, D, E, F, G, H, I, L, M, N, Q, R, S, T,

V, W, Y, preferably Y, W or H and at a position corresponding toposition 580 of SEQ ID NO: 2 there is an alteration to generate an aminoacid from the group consisting of C, D, E, F, G, H, I, L, M, N, P, R, S,T, V, W, Y, or

(b) at a position corresponding to position 573 of SEQ ID NO: 2 there isan alteration to generate a P and at a position corresponding toposition 580 of SEQ ID NO: 2 there is an alteration to generate a K andthe polypeptide comprises an additional alteration at a positionselected from the group consisting of 492, 550, and 574 (or equivalentpositions of other albumins or variants or fragment thereof).12. The polypeptide according to any preceding embodiment in which:(a) at a position corresponding to position 574 of SEQ ID NO: 2 there isan alteration to generate an amino acid from the group consisting of A,C, D, E, F, G, H, I, L, M, P, Q, R, S, T, V, W, Y, H, D, F, G, N, S orY, more preferably H, D, F or G, most preferably H and at a positioncorresponding to position 580 of SEQ ID NO: 2 there is an alteration togenerate at a position corresponding to position to an amino acid fromthe group consisting of A, C, D, E, F, G, H, I, L, M, N, P, R, S, T, V,W, Y, or(b) at a position corresponding to position 574 of SEQ ID NO: 2 there isan alteration to generate an N and at a position corresponding toposition 580 of SEQ ID NO: 2 there is an alteration to generate a K andthe polypeptide comprises an additional alteration at a positionselected from the group consisting of 492, 550, and 573 (or equivalentpositions of other albumins or variants or fragment thereof).13. The polypeptide according to any preceding embodiment, wherein thepolypeptide comprises alterations at three or more positions selectedfrom positions corresponding to positions 492, 550, 573, 574 and 580 inSEQ ID NO: 2.14. The polypeptide according to any preceding embodiment wherein thealteration at the position corresponding to position 492, 550, 573, 574and/or 580 is a substitution.15. The polypeptide of embodiment 14 wherein the substitution at theposition corresponding to position 492 is to G, D, F, H, M or R.16. The polypeptide of embodiment 14 or 15 wherein the substitution atthe position corresponding to position 492 is to G or D.17. The polypeptide of any of embodiments 13 to 15 wherein thesubstitution at the position corresponding to position 492 is to G.18. The polypeptide of any of embodiments 13 to 15 wherein thesubstitution at the position corresponding to position 492 is to D.19. The polypeptide of any of embodiments 13 to 18 wherein thesubstitution at the position corresponding to position 550 is to K, L, ME, or R.20. The polypeptide of any of embodiments 13 to 19 wherein thesubstitution at the position corresponding to position 550 is to K, L orM.21. The polypeptide of any of embodiments 13 to 20 wherein thesubstitution at the position corresponding to position 550 is to K.22. The polypeptide of any of embodiments 13 to 21 wherein thealteration at the position corresponding to position 573 is asubstitution to P, Y, W, H, F, T, I or V.23. The polypeptide of any of embodiments 13 to 22 wherein thesubstitution at the position corresponding to position 573 is to P, Y orW.24. The polypeptide of any of embodiments 13 to 23 wherein thesubstitution at the position corresponding to position 573 is to a P.25. The polypeptide of any of embodiments 13 to 24 wherein thealteration at the position corresponding to position 574 is asubstitution to H, G, D, F, N, S or Y.26. The polypeptide of any of embodiments 13 to 25 wherein thesubstitution at the position corresponding to position 574 is to D, F, Gor H.27. The polypeptide of any of embodiments 13 to 26 wherein thesubstitution at the position corresponding to position 574 is to H.28. The polypeptide of any of embodiments 13 to 27 wherein thesubstitution at the position corresponding to position 580 is to K or R.29. The polypeptide of any of embodiments 13 to 28 wherein thesubstitution at the position corresponding to position 580 is to K.30. The polypeptide of any preceding embodiment wherein the polypeptidecomprises alterations at positions corresponding to positions (i) 573and 580; (ii) 492 and 573; or (iii) 573 and 574 of SEQ ID NO: 2.31. The polypeptide of any preceding embodiment wherein the polypeptidecomprises alterations at two or more positions corresponding to thefollowing positions of SEQ ID NO: 2: 580K+573P (e.g. SEQ ID NO: 128);580R+573P (e.g. SEQ ID NO: 129); 574D+573P (e.g. SEQ ID NO: 121);574F+573P (e.g. SEQ ID NO: 122); 574G+573P (e.g. SEQ ID NO: 123);574H+573P (e.g. SEQ ID NO: 124); 574N+573P (e.g. SEQ ID NO: 125);574S+573P (e.g. SEQ ID NO: 126); 550K+580K (e.g. SEQ ID NO: 132);550K+574H (e.g. SEQ ID NO: 130); 550K+573P (e.g. SEQ ID NO: 117);492D+573P (e.g. SEQ ID NO: 108); 492F+573P (e.g. SEQ ID NO: 109);492H+573P (e.g. SEQ ID NO: 111); 492R+573P (e.g. SEQ ID NO: 112);574H+580K (e.g. SEQ ID NO: 134); 550L+574H (e.g. SEQ ID NO: 245);550L+580K (e.g. SEQ ID NO: 247); 550M+580K (e.g. SEQ ID NO: 251);492D+550K (e.g. SEQ ID NO: 231); 550M+574H (e.g. SEQ ID NO: 249);492D+574H (e.g. SEQ ID NO: 232); 492G+550K (e.g. SEQ ID NO: 240);550M+574H (e.g. SEQ ID NO: 249); or 492G+574H (e.g. SEQ ID NO: 241).32. The polypeptide according to any preceding embodiment wherein thepolypeptide comprises three or more alterations at positions selectedfrom the group consisting of positions corresponding to the followingpositions of SEQ ID NO: 2: 492, 550, 573, 574 and 580.33. The polypeptide of embodiment 32 wherein the polypeptide comprisesalterations at positions corresponding to the following positions of SEQID NO: 2: 574H+580K+573P (e.g. SEQ ID NO: 135); 550K+574H+573P (e.g. SEQID NO: 131); 492D+550K+573P (e.g. SEQ ID NO: 253); 550M+573P+580K (e.g.SEQ ID NO: 252); 550L+573P+580K (e.g. SEQ ID NO: 253); 492G+573P+580K(e.g. SEQ ID NO: 242); 550M+573P+574H (e.g. SEQ ID NO: 250);492G+550K+573P (e.g. SEQ ID NO: 254); 550L+573P+574H (e.g. SEQ ID NO:246); 492D+573P+580K (e.g. SEQ ID NO: 234); 492D+573P+574H (e.g. SEQ IDNO: 233); 492G+574H+580K (e.g. SEQ ID NO: 263); 492G+550K+580K (e.g. SEQID NO: 259); 492D+550K+580K (e.g. SEQ ID NO: 258); 492D+574H+580K (e.g.SEQ ID NO: 262); or 492G+550K+574H (e.g. SEQ ID NO: 255).34. The polypeptide according to any preceding embodiment wherein thepolypeptide comprises four or more alterations at positions selectedfrom the group consisting of positions corresponding to the followingpositions of SEQ ID NO: 2: 492, 550, 573, 574 and 580.35. The polypeptide of embodiment 34 wherein the polypeptide comprisesalterations at positions corresponding to the following positions of SEQID NO: 2: 492G+573P+574H+580K (e.g. SEQ ID NO: 115); 492D+573P+574H+580K(e.g. SEQ ID NO: 114); 550K+573P+574H+580K (e.g. SEQ ID NO: 265);492G+550K+573P+580K (e.g. SEQ ID NO: 261); 492F+573P+574H+580K (e.g. SEQID NO: 237); 492G+573P+574G+580K (e.g. SEQ ID NO: 243);492D+573P+574H+580R (e.g. SEQ ID NO: 236); 492G+573P+574H+580R (e.g. SEQID NO: 244); 492D+550K+573P+580K (e.g. SEQ ID NO: 260);492D+550K+573P+574H (e.g. SEQ ID NO: 256); 492F+573P+574H+580R (e.g. SEQID NO: 239); 492D+573P+574G+580K (e.g. SEQ ID NO: 235); or492F+573P+574G+580K (e.g. SEQ ID NO: 238);36. The polypeptide according to any preceding embodiment wherein thepolypeptide comprises five or more alterations at positions selectedfrom the group consisting of positions corresponding to the followingpositions of SEQ ID NO: 2: 492, 550, 573, 574 and 580.37. The polypeptide according to embodiment wherein the polypeptidecomprises alterations at positions corresponding to the followingpositions of SEQ ID NO: 2: 492G+550K+573P+574H+580K (e.g. SEQ ID NO:267) or 492D+550K+573P+574H+580K (e.g. SEQ ID NO: 266).38. The polypeptide according to any preceding embodiment comprisingalterations corresponding to the following positions in SEQ ID NO: 2:492G+573P+574H+580K (e.g. SEQ ID NO: 115); 492G+550K+573P+574H (e.g. SEQID NO: 257); 492D+550K+573P+574H (e.g. SEQ ID NO: 256); 492G+550K+573P(e.g. SEQ ID NO: 254); 492D+550K+573P (e.g. SEQ ID NO: 253);550M+573P+580K (e.g. SEQ ID NO: 252); 550L+573P+580K (e.g. SEQ ID NO:248); 550L+573P+574H (e.g. SEQ ID NO: 246); 492G+573P+580K (e.g. SEQ IDNO: 242); 492D+573P+580K (e.g. SEQ ID NO: 234); 573P+574H+580K (e.g. SEQID NO: 135); 550K+573P+580K (e.g. SEQ ID NO: 133); 550K+573P+574H (e.g.SEQ ID NO: 131); or 492D+573P+574H+580K (SEQ ID NO: 114).39. The polypeptide of any of embodiments 1 to 38 wherein the referencealbumin is HSA (SEQ ID No: 2) or a fragment thereof, or a fusionpolypeptide comprising HSA or a fragment thereof, most preferably SEQ IDNO: 2.40. The polypeptide according to any of embodiments 1 to 39, having astronger binding affinity to FcRn and/or longer plasma half-life than aparent albumin, reference albumin, fragment thereof or fusionpolypeptide comprising said parent albumin, reference albumin orfragment or fusion thereof.41. The polypeptide according any of embodiments 1 to 40, wherein thesequence identity of the polypeptide to SEQ ID NO: 2 is more than 80%,preferably more than 90%, more preferred more than 95%, more preferredmore than 96%, even more preferred more than 97%, more preferred morethan 98% and most preferred more than 99%.42. A fusion polypeptide comprising a polypeptide according to any ofembodiments 1 to 41 and a fusion partner polypeptide selected from atherapeutic, prophylactic, diagnostic, imaging or other beneficialmoiety.43. A method for preparing a polypeptide which is a variant of albumin,fragment thereof or fusion polypeptide comprising said variant albuminor fragment thereof having a binding affinity to FcRn which is alteredcompared to the binding affinity of a reference albumin, fragment orfusion thereof to FcRn, comprising:

(a) Providing a nucleic acid encoding a parent albumin having at least80% sequence identity to SEQ ID NO: 2;

(b) Modifying the sequence of step (a), to encode a polypeptide which isa variant albumin, fragment thereof or fusion polypeptide comprisingsaid variant albumin or fragment thereof comprising alterations at twoor more positions corresponding to positions selected from 492, 550,573, 574 and 580 in SEQ ID NO: 2, preferably as described in any ofembodiments 1 to 13;

(c) Introducing the modified sequence of step (b) in a suitable hostcell;

(d) Growing the cells in a suitable growth medium under conditionleading to expression of the polypeptide; and

(e) Recovering the polypeptide from the growth medium;

wherein the polypeptide has an altered binding affinity to FcRn and/oran altered plasma half-life compared with the half-life of a parentalbumin, reference albumin, fragment thereof or fusion polypeptidecomprising said parent albumin, reference albumin or fragment or fusionthereof.

44. The method of embodiment 43 wherein the substitution at the positioncorresponding to position 492 is a substitution to G, D, F, H, M or R,preferably G or D45. The method of embodiment 44 wherein the substitution at the positioncorresponding to position 492 is to G.46. The method of embodiment 44 wherein the substitution at the positioncorresponding to position 492 is to D.47. The method of any of embodiments 43 to 46 wherein the substitutionat the position corresponding to position 550 is to K, L, M, E or R,preferably K, L or M, most preferably K.48. The method of any of embodiments 43 to 47 wherein the substitutionat the position corresponding to position 573 is a substitution to P, Y,W, H, F, T, I or V, preferably P, Y, or W, most preferably P.49. The method of any of embodiments 43 to 48 wherein the substitutionat the position corresponding to position 574 is a substitution to H, D,F, G, N, S or Y, preferably H, D, F, or G, most preferably H.50. The method of any of embodiments any of embodiments 43 to 49 whereinthe substitution at the position corresponding to position 580 is asubstitution to K or R, most preferably R.51. The method of any of embodiments 43 to 50 wherein the two or morealteraions are at positions corresponding to positions (i) 573 and 580;(ii) 492 and 573; or (iii) 573 and 574 of SEQ ID NO: 2.52. The method according to any of embodiments 43 to 51 wherein the twoor more alterations are at positions corresponding to the followingpositions of SEQ ID NO: 2: 580K+573P (e.g. SEQ ID NO: 128); 580R+573P(e.g. SEQ ID NO: 129); 574D+573P (e.g. SEQ ID NO: 121); 574F+573P (e.g.SEQ ID NO: 122); 574G+573P (e.g. SEQ ID NO: 123); 574H+573P (e.g. SEQ IDNO: 124); 574N+573P (e.g. SEQ ID NO: 125); 574S+573P (e.g. SEQ ID NO:126); 550K+580K (e.g. SEQ ID NO: 132); 550K+574H (e.g. SEQ ID NO: 130);550K+573P (e.g. SEQ ID NO: 117); 492D+573P (e.g. SEQ ID NO: 108);492F+573P (e.g. SEQ ID NO: 109); 492H+573P (e.g. SEQ ID NO: 111);492R+573P (e.g. SEQ ID NO: 112); 574H+580K (e.g. SEQ ID NO: 134);550L+574H (e.g. SEQ ID NO: 245); 550L+580K (e.g. SEQ ID NO: 247);550M+580K (e.g. SEQ ID NO: 251); 492D+550K (e.g. SEQ ID NO: 231);550M+574H (e.g. SEQ ID NO: 249); 492D+574H (e.g. SEQ ID NO: 232);492G+550K (e.g. SEQ ID NO: 240); 550M+574H (e.g. SEQ ID NO: 249); or492G+574H (e.g. SEQ ID NO: 241).53. The method according to any of embodiments 43 to 52 comprising threeor more alterations at positions selected from the group consisting ofpositions corresponding to the following positions of SEQ ID NO: 2: 492,550, 573, 574 and 580.54. The method according to any of embodiments 43 to 53 comprisingalterations at positions corresponding to the following positions of SEQID NO: 2: 574H+580K+573P (e.g. SEQ ID NO: 135); 550K+574H+573P (e.g. SEQID NO: 131); 492D+550K+573P (e.g. SEQ ID NO: 253); 550M+573P+580K (e.g.SEQ ID NO: 252); 550L+573P+580K (e.g. SEQ ID NO: 253); 492G+573P+580K(e.g. SEQ ID NO: 242); 550M+573P+574H (e.g. SEQ ID NO: 250);492G+550K+573P (e.g. SEQ ID NO: 254); 550L+573P+574H (e.g. SEQ ID NO:246); 492D+573P+580K (e.g. SEQ ID NO: 234); 492D+573P+574H (e.g. SEQ IDNO: 233); 492G+574H+580K (e.g. SEQ ID NO: 263); 492G+550K+580K (e.g. SEQID NO: 259); 492D+550K+580K (e.g. SEQ ID NO: 258); 492D+574H+580K (e.g.SEQ ID NO: 262); or 492G+550K+574H (e.g. SEQ ID NO: 255).55. The method according to any of embodiments 43 to 54 comprising fouror more alterations at positions selected from the group consisting ofpositions corresponding to the following positions of SEQ ID NO: 2: 492,550, 573, 574 and 580.56. The method according to any of embodiments 43 to 55 comprisingalterations at positions corresponding to the following positions of SEQID NO: 2: 492G+573P+574H+580K (e.g. SEQ ID NO: 115); 492D+573P+574H+580K(e.g. SEQ ID NO: 114); 550K+573P+574H+580K (e.g. SEQ ID NO: 265);492G+550K+573P+580K (e.g. SEQ ID NO: 261); 492F+573P+574H+580K (e.g. SEQID NO: 237); 492G+573P+574G+580K (e.g. SEQ ID NO: 243);492D+573P+574H+580R (e.g. SEQ ID NO: 236); 492G+573P+574H+580R (e.g. SEQID NO: 244); 492D+550K+573P+580K (e.g. SEQ ID NO: 260);492D+550K+573P+574H (e.g. SEQ ID NO: 256); 492F+573P+574H+580R (e.g. SEQID NO: 239); 492D+573P+574G+580K (e.g. SEQ ID NO: 235); or492F+573P+574G+580K (e.g. SEQ ID NO: 238);57. The method according to any of embodiments 43 to 56 comprising fiveor more alterations at positions selected from the group consisting ofpositions corresponding to the following positions of SEQ ID NO: 2: 492,550, 573, 574 and 580.58. The polypeptide according to any of embodiments 43 to 57 comprisingalterations at positions corresponding to the following positions of SEQID NO: 2: 492G+550K+573P+574H+580K (e.g. SEQ ID NO: 267) or492D+550K+573P+574H+580K (e.g. SEQ ID NO: 266).59. The method according to any of embodiments 43 to 58 comprisingalterations at positions corresponding to the following positions in SEQID NO: 2: 492G+573P+574H+580K (e.g. SEQ ID NO: 115); 492G+550K+573P+574H(e.g. SEQ ID NO: 257); 492D+550K+573P+574H (e.g. SEQ ID NO: 256);492G+550K+573P (e.g. SEQ ID NO: 254); 492D+550K+573P (e.g. SEQ ID NO:253); 550M+573P+580K (e.g. SEQ ID NO: 252); 550L+573P+580K (e.g. SEQ IDNO: 248); 550L+573P+574H (e.g. SEQ ID NO: 246); 492G+573P+580K (e.g. SEQID NO: 242); 492D+573P+580K (e.g. SEQ ID NO: 234); 573P+574H+580K (e.g.SEQ ID NO: 135); 550K+573P+580K (e.g. SEQ ID NO: 133); 550K+573P+574H(e.g. SEQ ID NO: 131); or 492D+573P+574H+580K (SEQ ID NO: 114).60. The method any of embodiments 43 to 59 wherein the reference albuminis HSA (SEQ ID No: 2) or a fragment thereof, or a fusion polypeptidecomprising HSA or a fragment thereof, most preferably SEQ ID NO: 2.61. The method according any of embodiments 43 to 60, wherein thesequence identity of the polypeptide to SEQ ID NO: 2 is more than 80%,preferably more than 90%, more preferred more than 95%, more preferredmore than 96%, even more preferred more than 97%, more preferred morethan 98% and most preferred more than 99%.62. A conjugate comprising a polypeptide according to any of embodiments1 to 42 or obtainable by a method according to any of embodiments 43 to61 and a conjugation partner.63. The conjugate according to embodiment 62 wherein the conjugationpartner is a therapeutic, prophylactic, diagnostic, imaging or otherbeneficial moiety.64. An associate comprising a polypeptide according to any ofembodiments 1 to 42 or obtainable by a method according to any ofembodiments 43 to 61 and a therapeutic, prophylactic, diagnostic,imaging or other beneficial moiety.65. A nanoparticle or microparticle comprising a polypeptide accordingto any of embodiments 1 to 42 or obtainable by a method according to anyof embodiments 43 to 61, a conjugate according to embodiment 62 or 63 oran associate according to embodiment 64.66. A composition comprising a polypeptide according to any ofembodiments 1 to 42 or obtainable by a method according to any ofembodiments 43 to 61, a conjugate according to embodiment 62 or 63, anassociate according to embodiment 64 or a nanoparticle or microparticleaccording embodiment 65, wherein the binding affinity of thepolypeptide, fusion polypeptide, conjugate, associate or nanoparticle ormicroparticle to FcRn is stronger than the binding affinity of acomposition comprising the corresponding parent albumin, referencealbumin, fragment thereof or fusion polypeptide, conjugate, associate ornanoparticle or microparticle comprising said parent albumin, referencealbumin or fragment or fusion thereof to FcRn.67. A composition according to embodiment 66 where the binding affinityof the polypeptide, fusion polypeptide, conjugate, associate ornanoparticle or microparticle to FcRn is stronger than the bindingaffinity of HSA to FcRn.68. A composition according to embodiment 66 or 67, wherein the bindingcoefficient of the variant of the polypeptide, fusion polypeptide,conjugate, associate or nanoparticle or microparticle to FcRn is lessthan 0.9×KD of HSA to FcRn, more preferred less than 0.5×KD of HSA toFcRn, more preferred less than 0.1×KD of HSA to FcRn, even morepreferred less than 0.05×KD of HSA to FcRn, even more preferred lessthan 0.02×KD of HSA to FcRn and most preferred less than 0.01×KD of HSAto FcRn.69. The composition according to any of embodiments 66 to 68, comprisinga polypeptide according to any of embodiments 1 to 42 or obtainable by amethod according to any of embodiments 43 to 61, a conjugate accordingto embodiment 62 or 63, an associate according to embodiment 64 or ananoparticle or microparticle according embodiment 65, furthercomprising a compound comprising an antibody binding domain (ABD) and atherapeutic, prophylactic, diagnostic, imaging or other beneficialmoiety.70. The composition according to any of embodiments 66 to 69, comprisinga pharmaceutically acceptable carrier or excipient.71. Use of a polypeptide according to any of embodiments 1 to 42 orobtainable by a method according to any of embodiments 43 to 61, aconjugate according to embodiment 62 or 63, an associate according toembodiment 64 or a nanoparticle or microparticle according embodiment 65or a composition according to any of embodiments 66 to 70 to alter thebinding affinity to FcRn or half-life, preferably in plasma, of atherapeutic, prophylactic, diagnostic, imaging or other beneficialmoiety.72. The use according to embodiment 71 wherein the binding affinity toFcRn is increased relative to the binding affinity of a referencecomprising or consisting of HSA (SEQ ID NO: 2) or a fragment, fusion,conjugate, associate, nanoparticle or microparticle thereof to FcRn.73. The use according to embodiment 71 wherein the binding affinity toFcRn is decreased relative to the binding affinity of a referencecomprising or consisting of HSA (SEQ ID NO: 2) or a fragment, fusion,conjugate, associate, nanoparticle or microparticle thereof to FcRn.74. A method for altering the binding affinity to FcRn or half-lifepreferably in plasma, of a molecule comprising:

(a) where the molecule is a polypeptide, fusing or conjugating themolecule to a polypeptide according to any of embodiments 1 to 42 orobtainable by a method of embodiments 43 to 61, or to a conjugateaccording to embodiment 62 or 63; associating the molecule to apolypeptide according to any of embodiments 1 to 42 or obtainable by amethod of embodiments 43 to 61 or to a conjugate according to embodiment62 or 63; incorporating the molecule in an associate according toembodiment 64, in nanoparticle or microparticle according to embodiment65 or a composition according to any of embodiments 66 to 70;

(b) where the molecule is not a polypeptide, conjugating the molecule toa polypeptide according to any of embodiments 1 to 42 or obtainable by amethod of embodiments 43 to 61, or to a conjugate according toembodiment 62 or 63; associating the molecule to a polypeptide accordingto any of embodiments 1 to 42 or obtainable by a method of embodiments43 to 61 or to a conjugate according to embodiment 62 or 63;incorporating the molecule in an associate according to embodiment 64,in nanoparticle or microparticle according to embodiment 65 or acomposition according to any of embodiments 66 to 70.

75. A method according to embodiment 74 wherein the molecule is atherapeutic, prophylactic, diagnostic, imaging or other beneficialmoiety.76. A polypeptide, fusion polypeptide, conjugate, associate,nanoparticle or microparticle or composition thereof according to any ofembodiments 1 to 42 or 62 to 70 or obtainable by the method ofembodiments 43 to 61 wherein the polypeptide, fusion polypeptide,conjugate, associate, nanoparticle or microparticle or compositioncomprises one or more (several) moiety selected from those describedherein.77. A nucleic acid encoding the polypeptide or fusion polypeptide of anyof embodiments 1 to 76.78. A vector comprising a nucleic acid according to embodiment 77.79. A host cell comprising a nucleic acid according to embodiment 77 ora vector according to embodiment 78.80. A host cell according to embodiment 79 wherein the host cell is aeukaryote, preferably a yeast (such as Saccharomyces cerevisiae) or amammalian cell (such as CHO or HEK) or a plant cell (such as rice).81. A method of prophylaxis, treatment or diagnosis comprisingadministering a polypeptide, fusion polypeptide, conjugate, composition,associate, nanoparticle or microparticle or polynucleotide according toany of embodiments 1 to 42 or 62 to 70 or obtainable by the method ofany of embodiments 43 to 61 to a subject.

The invention is further described by the following examples that shouldnot be construed as limiting the scope of the invention.

EXAMPLES Example 1 Preparation of HSA variant expression plasmids forHSA

HSA variants were expressed using standard molecular biology techniques,such as described in Sambrook, J. and D. W. Russell, 2001 (MolecularCloning: a laboratory manual, 3r^(d) ed. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y).

Method 1:

Permutation libraries at positions E492, D550, K574H and Q580K wereproduced by PCR amplification of pDB4081, encoding wild type HSA(described below), using a mutagenic forward primer and non-mutagenicreverse primer as shown in Table 2. The PCR conditions are shown inTables 3 and 4. 2 μl of reaction product was assessed by agarose gelelectrophoresis and the remainder treated with 5 μl of 10× buffer 4 (NewEngland Biolabs—50 mM potassium acetate, 20 mM Tris-acetate, 10 mMmagnesium acetate, 1 mM DTT pH 7.9 at 25° C.) and 1 μl DpnI (NEB) for 1hour at 37° C. The reactions were purified by addition of 50 μl water,prior to application of a vacuum for 20 minutes. A further 50 μl ofwater was added and a vacuum applied until the well was dry. Plasmid DNAwas recovered by addition of 30 μl of water and incubation for 1 minute.2 μl of this purified product was transformed into 15 μl TOP10 E. colicells by standard protocols. Clones were selected and grown overnight inLB supplemented with 100 μg/ml ampicillin and purified using a QiagenQIAprep 96 miniprep kit according to manufacturer's instructions.Variants were verified by sequencing.

pDB4081 was made by the ligation of a synthetic DNA fragment, Bsal/SphIdigested, which had been generated by gene assembly (DNA2.0 Inc, USA),containing 3′ region of the PRB1 promoter, modified fusion leadersequence, nucleotide sequence encoding HSA and 5′ region of the modifiedADH1 terminator) into HindIII/SphI-digested pDB4005. pDB4005 isdescribed in WO 2011/051489 (incorporated herein by reference).

TABLE 2 Plasmid and amino acid substitution and relevant primers VariantSEQ ID Oligo 1 Oligo 2 Plasmid Variant No. (SEQ ID No.) (SEQ ID No.)name HSA E492A 32 HSA-1 (136) REV E492 (212) pDB4768 HSA E492C 33 HSA-2(137) REV E492 (212) pDB4769 HSA E492D 34 HSA-3 (138) REV E492 (212)pDB4770 HSA E492F 35 HSA-4 (139) REV E492 (212) pDB4771 HSA E492G 36HSA-5 (140) REV E492 (212) pDB4772 HSA E492H 37 HSA-6 (141) REV E492(212) pDB4773 HSA E492I 38 HSA-7 (142) REV E492 (212) pDB4774 HSA E492K39 HSA-8 (143) REV E492 (212) pDB4775 HSA E492L 40 HSA-9 (144) REV E492(212) pDB4776 HSA E492M 41 HSA-10 (145) REV E492 (212) pDB4777 HSA E492N42 HSA-11 (146) REV E492 (212) pDB4778 HSA E492P 43 HSA-12 (147) REVE492 (212) pDB4779 HSA E492Q 44 HSA-13 (148) REV E492 (212) pDB4780 HSAE492R 45 HSA-14 (149) REV E492 (212) pDB4781 HSA E492S 46 HSA-15 (150)REV E492 (212) pDB4782 HSA E492T 47 HSA-16 (151) REV E492 (212) pDB4783HSA E492V 48 HSA-17 (152) REV E492 (212) pDB4784 HSA E492W 49 HSA-18(153) REV E492 (212) pDB4785 HSA E492Y 50 HSA-19 (154) REV E492 (212)pDB4856 HSA D550A 51 HSA-20 (155) REV D550 (213) pDB4786 HSA D550C 52HSA-21 (156) REV D550 (213) pDB4857 HSA D550E 53 HSA-22 (157) REV D550(213) pDB4858 HSA D550F 54 HSA-23 (158) REV D550 (213) pDB4859 HSA D550G55 HSA-24 (159) REV D550 (213) pDB4787 HSA D550H 56 HSA-25 (160) REVD550 (213) pDB4788 HSA D550I 57 HSA-26 (161) REV D550 (213) pDB4789 HSAD550K 58 HSA-27 (162) REV D550 (213) pDB4790 HSA D550L 59 HSA-28 (163)REV D550 (213) pDB4791 HSA D550M 60 HSA-29 (164) REV D550 (213) pDB4792HSA D550N 61 HSA-30 (165) REV D550 (213) pDB4793 HSA D550P 62 HSA-31(166) REV D550 (213) pDB4794 HSA D550Q 63 HSA-32 (167) REV D550 (213)pDB4795 HSA D550R 64 HSA-33 (168) REV D550 (213) pDB4796 HSA D550S 65HSA-34 (169) REV D550 (213) pDB4797 HSA D550T 66 HSA-35 (170) REV D550(213) pDB4798 HSA D550V 67 HSA-36 (171) REV D550 (213) pDB4799 HSA D550Y68 HSA-37 (172) REV D550 (213) pDB4800 HSA D550W 69 HSA-38 (173) REVD550 (213) pDB4801 HSA K574A 70 HSA-39 (174) REV K574 (214) pDB4802 HSAK574C 71 HSA-40 (175) REV K574 (214) pDB4803 HSA K574D 72 HSA-41 (176)REV K574 (214) pDB4804 HSA K574E 73 HSA-42 (177) REV K574 (214) pDB4805HSA K574F 74 HSA-43 (178) REV K574 (214) pDB4806 HSA K574G 75 HSA-44(179) REV K574 (214) pDB4807 HSA K574H 76 HSA-45 (180) REV K574 (214)pDB4808 HSA K574I 77 HSA-46 (181) REV K574 (214) pDB4809 HSA K574L 78HSA-47 (182) REV K574 (214) pDB4810 HSA K574M 79 HSA-48 (183) REV K574(214) pDB4811 HSA K574N 80 HSA-49 (184) REV K574 (214) pDB4812 HSA K574P81 HSA-50 (185) REV K574 (214) pDB4813 HSA K574Q 82 HSA-51 (186) REVK574 (214) pDB4814 HSA K574R 83 HSA-52 (187) REV K574 (214) pDB4815 HSAK574S 84 HSA-53 (188) REV K574 (214) pDB4816 HSA K574T 85 HSA-54 (189)REV K574 (214) pDB4817 HSA K574V 86 HSA-55 (190) REV K574 (214) pDB4818HSA K574Y 87 HSA-56 (191) REV K574 (214) pDB4819 HSA K574W 88 HSA-57(192) REV K574 (214) pDB4820 HSA Q580A 89 HSA-58 (193) REV Q580 (215)pDB4821 HSA Q580C 90 HSA-59 (194) REV Q580 (215) pDB4822 HSA Q580D 91HSA-60 (195) REV Q580 (215) pDB4823 HSA Q580E 92 HSA-61 (196) REV Q580(215) pDB4824 HSA Q580F 93 HSA-62 (197) REV Q580 (215) pDB4825 HSA Q580G94 HSA-63 (198) REV Q580 (215) pDB4826 HSA Q580H 95 HSA-64 (199) REVQ580 (215) pDB4827 HSA Q580I 96 HSA-65 (200) REV Q580 (215) pDB4828 HSAQ580K 97 HSA-66 (201) REV Q580 (215) pDB4829 HSA Q580L 98 HSA-67 (202)REV Q580 (215) pDB4830 HSA Q580M 99 HSA-68 (203) REV Q580 (215) pDB4831HSA Q580N 100 HSA-69 (204) REV Q580 (215) pDB4832 HSA Q580P 101 HSA-70(205) REV Q580 (215) pDB4833 HSA Q580R 102 HSA-71 (206) REV Q580 (215)pDB4834 HSA Q580S 103 HSA-72 (207) REV Q580 (215) pDB4835 HSA Q580T 104HSA-73 (208) REV Q580 (215) pDB4836 HSA Q580V 105 HSA-74 (209) REV Q580(215) pDB4837 HSA Q580Y 106 HSA-75 (210) REV Q580 (215) pDB4838 HSAQ580W 107 HSA-76 (211) REV Q580 (215) pDB4839

TABLE 3 PCR reaction components *HF buffer (5x) 10 μl  template DNA (20ng/μl)   1 μl dNTP (2.5 mM) 5 μl *Phusion (polymerase)  0.5 μl oligo 1(20 μM) 1 μl dH₂O 31.5 μl oligo 2 (20 μM) 1 μl (*HF buffer and Phusionpolymerase are from New England Biolabs)

TABLE 4 PCR reaction conditions Temperature Cycle length Number ofcycles 98° C.  3 min 1 98° C. 15 sec 30 65° C. 35 sec 72° C. 11 min 72°C. 11 min 1

Method 2:

Variants described in Table 5 and 6 were produced as described inWO2012/150319, Example 6, Method 2 ‘Production of combination Variantswith K573P’ (incorporated herein by reference), with the followingmodifications. For variants shown in Table 5, a fragment encoding theK573P mutation was removed from pDB4673 via the SalI and Bsu361restriction sites and inserted into similarly digested parent plasmid,as indicated in Table 5. pDB4673 (HSA K573P) was constructed byinsertion of the fragment produced by digestion of pD4283 (described inWO 2011/051489, incorporated herein by reference) with SalI and HinDIIIrestriction enzymes into similarly digested pDB4081. For variants shownin Table 6, the fragment encoding K573P+K574H+Q580K was removed frompDB5032 using the SalI and Bsu36I restriction sites and inserted intosimilarly digested parent plasmid, as indicated in Table 6.

TABLE 5 Plasmid and amino acid substitution Variant Relevant SEQ IDParent Plasmid Variant No. Plasmid name HSA E492D + K573P 108 pDB4770pDB4990 HSA E492F + K573P 109 pDB4771 pDB4991 HSA E492G + K573P 110pDB4772 pDB4992 HSA E492H + K573P 111 pDB4773 pDB4993 HSA E492M + K573P112 pDB4777 pDB4994 HSA E492R + K573P 113 pDB4781 pDB4995

TABLE 6 Plasmid and amino acid substitution Relevant Variant SEQ ParentPlasmid Variant ID No. Plasmid name HSA E492D + K573P + 114 pDB4770pDB5091 K574H + Q580K HSA E492G + K573P + 115 pDB4772 pDB5092 K574H +Q580K

Method 3:

Variants described in Table 7 were produced as described inWO2012/150319, Example 6, Method 1 (incorporated herein by reference),using the plasmids indicated in the tables as templates for the PCRreactions. The conditions described in Tables 8 and 9 were used toproduce pDB4997-5004, pDB 5042-46, and pDB5030-1. Those described inTables 10 and 11 were used to produce pDB5027-29 and pDB5032. Themutated PCR products were digested using SalI and Bsu361 restrictionenzymes and inserted into similarly digested pDB4081, encoding wild typeHSA (as described above).

TABLE 7 Plasmid and amino acid substitution and relevant primers VariantOligo 1 SEQ ID (SEQ ID Oligo 2 Template Plasmid Variant No. No.) (SEQ IDNo.) plasmid Name HSA D550E + K573P 116 KBf4 (216) KBr30 (217) pDB4858pDB5042 HSA D550K + K573P 117 KBf4 (216) KBr30 (217) pDB4790 pDB5043 HSAD550L + K573P 118 KBf4 (216) KBr30 (217) pDB4791 pDB5044 HSA D550M +K573P 119 KBf4 (216) KBr30 (217) pDB4792 pDB5045 HSA D550R + K573P 120KBf4 (216) KBr30 (217) pDB4796 pDB5046 HSA K574D + K573P 121 KBf4 (216)KBr21 (218) pDB4673 pDB4997 HSA K574F + K573P 122 KBf4 (216) KBr22 (219)pDB4673 pDB4998 HSA K574G + K573P 123 KBf4 (216) KBr23 (220) pDB4673pDB4999 HSA K574H + K573P 124 KBf4 (216) KBr24 (221) pDB4673 pDB5000 HSAK574N + K573P 125 KBf4 (216) KBr25 (222) pDB4673 pDB5001 HSA K574S +K573P 126 KBf4 (216) KBr26 (223) pDB4673 pDB5002 HSA K574Y + K573P 127KBf4 (216) KBr27 (224) pDB4673 pDB5005 HSA Q580K + K573P 128 KBf4 (216)KBr28 (225) pDB4673 pDB5003 HSA Q580R + K573P 129 KBf4 (216) KBr29 (226)pDB4673 pDB5004 HSA D550K + K574H 130 KBf4 (216) KBr31 (227) pDB4790pDB5027 HSA 131 KBf4 (216) KBr24 (221) pDB4790 pDB5028 D550K + K574H +K573P HSA D550K + Q580K 132 KBf4 (216) KBr32 (228) pDB4790 pDB5029 HSA133 KBf4 (216) KBr28 (225) pDB5043 pDB5030 D550K + Q580K + K573P HSAK574H + Q580K 134 KBf4 (216) KBr33 (229) pDB4808 pDB5031 HSA 135 KBf4(216) KBr34 (230) pDB5003 pDB5032 K574H + Q580K + K573P

TABLE 8 PCR reaction components HF buffer (5x) 10 μl dNTP (10 mM) 1 μloligo 1 (10 μM) 1 μl oligo 2 (10 μM) 1 μl template DNA (5 ng/μl) 1 μlPhusion (polymerase) 0.5 μl dH₂O 35.5 μl

TABLE 9 PCR reaction conditions Temperature Cycle length Number ofcycles 98° C. 2 min 1 98° C. 10 sec 35 61° C. 20 sec 72° C. 30 sec 72°C. 7 min 1

TABLE 10 PCR reaction components HF buffer (5x) 20 μl  dNTP (10 mM) 2 μloligo 1 (10 μM) 2 μl oligo 2 (10 μM) 2 μl template DNA (5 ng/μl) 2 μlPhusion (polymerase) 1 μl dH₂O 71 μl 

TABLE 11 PCR reaction conditions Temperature Cycle length Number ofcycles 98° C. 2 min 1 98° C. 10 sec 35 61° C. 30 sec 72° C. 20 sec 72°C. 10 min 1

Preparation of expression plasmids and transformation of S. cerevisiaewas performed as described in WO2012/150319 (incorporated herein byreference), employing the 24 (pDB5027-9, pDB5032 and pDB5091-92), or 48hour stocking method (all remaining variants). The host strain forpDB4990-5, pDB4997-5004, pDB5042-5046, pDB5027-32 and pDB5091-2 was S.cerevisiae DYB7 (Payne et al (2008) Applied and EnvironmentalMicrobiology Vol 74(24): 7759-7766) with four copies of PDI integratedinto the genome. The host strain for the remaining plasmids was S.cerevisiae BXP10cir° as described in WO2012/150319 (incorporated hereinby reference). Protein isolation and purification from shake flask wasperformed as described in WO2011/051489.

Example 2 SPR Analysis of Binding Affinity of WT HSA and Variants toshFcRn

SPR analyses were performed on a Biacore 3000 instrument (GEHealthcare). Immobilisation was carried out on CM5 chips coupled withHis-tagged shFcRn (FcRn produced by GeneArt, Life Technologies) using GEHealthcare amine coupling chemistry as per the manufacturer'sinstructions. Immobilised levels of shFcRn-HIS (shFcRn with a 6-His tailon the C-terminus of beta-2-microglobulin) were 1200-2500RU and achievedby injecting 20 μg/mL shFcRn diluted using sodium acetate pH4.5 (G EHealthcare). Chip surface was left to stabilize with a constant flow (5μL/min) of running buffer—Di-basic/Mono-basic phosphate buffer pH5.5((67 mM phosphate buffer, 0.15 M NaCl, 0.005% Tween 20) at pH 5.5)) at25° C. (i.e. ambient temperature) overnight. After ligand stabilization,the chip surface was conditioned by injecting 5−12×45 μLDi-basic/Mono-basic phosphate buffer at 30 μL/min followed by HBS_EP(0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20) at pH 7.4(GE Healthcare)) regeneration steps (12s) in between each injection.Surfaces were then checked for activity by injecting 3×45 μL positivecontrol (wt HSA (SEQ ID NO: 2)) at 30 μL/min, followed by 12sregeneration pulse. Kinetic measurements were performed by injectingdilutions (100 μM-0.016 μM) of HSA and HSA variants at 30 μL/min overimmobilised shFcRn, at 25° C. (i.e. ambient temperature). The referencecell value was then subtracted and Biaevaluation software 4.1 used toobtain kinetic data and confirm KD values. The variants were wild-typealbumin (SEQ ID NO: 2) and albumins with alterations at one or more ofpositions 492, 550, 573, 574 and 580. The variants were analysed by SPRto determine their binding response (RU) to shFcRn. Some variants werefurther characterized to determine KD values. The data are shown inTables 12 to 18.

TABLE 12 Binding affinity of selected albumin variants (with a singlealteration) to shFcRn Binding % increase in SEQ ID response bindingaffinity Run Analyte No. (RU) compared to WT A WT HSA 2 157 — HSA-K573P3 247 57.3 HSA-E492D 34 192 22.5 HSA-E492F 35 200 27.6 HSA-E492G 36 18719.4 HSA-E492H 37 186 18.8 HSA-E492M 41 184 17.5 HSA-E492R 45 184 17.5 BWT HSA 2 150 — HSA-K573P 3 211 40.7 HSA-D550E 53 175 16.8 HSA-D550K 58186 24.4 HSA-D550L 59 187 24.6 HSA-D550M 60 177 18.0 HSA-D550R 64 17718.2 C WT HSA 2 171 — HSA-K573P 3 223 30.4 HSA-K574D 72 214 25.4HSA-K474F 74 219 27.1 HSA-K574G 75 225 31.7 HSA-K574H 76 230 34.7HSA-K574N 80 205 20.4 HSA-K574S 84 205 20.1 HSA-K574Y 87 207 21.1 D WTHSA 2 159 — HSA-K573P 3 227 42.8 HSA-Q580K 97 212 33.4 HSA-Q580R 102 19522.3

The data in Table 12 show that variants comprising an alteration at oneof positions 492, 550, 574 and 580 show a higher FcRn binding affinitythan WT albumin.

TABLE 13 Binding affinity of albumin variants (with two alterations) toshFcRn Binding % increase in SEQ ID response binding affinity AnalyteNo. (RU) compared to WT WT HSA 2 242.3 — HSA-K573P 3 278.0 14.7HSA-E492D + K573P 108 270.8 11.8 HSA-E492F + K573P 109 270.3 11.6HSA-E492G + K573P 110 275.7 13.8

The data in Table 13 show that variants comprising alterations atposition 492 in conjunction with the K573P variant show higher bindingaffinity than WT albumin.

TABLE 14 Binding affinity of albumin variants to shFcRn Fold SEQ Ka KdMean difference ID (10³/ (10⁻³/ KD KD from Analyte NO: Ms) s) (μM) (μM)HSA-WT WT HSA (run 1) 2 10.9 40 3.67 4.02 — WT HSA (run 2) 2 9.15 39.94.36 HSA-K573P (run 1) 3 5.99 2.15 0.358 0.39 10.3 HSA-K573P (run 2) 35.95 2.53 0.426 HSA-E492D + 108 11.5 1.68 0.147 0.162 24.8 K573P (run 1)HSA-E492D + 108 11.1 1.96 0.177 K573P (run 2) HSA-E492G + 110 12.7 2.150.17 0.175 23.0 K573P (run 1) HSA-E492G + 110 11.3 2.04 0.18 K573P (run2)

Variants HSA-E492D+K573P and HSA-E492G+K573P were selected for furtheranalysis based on their apparent slow ‘off rates’ (i.e. dissociationconstants (Kd)). The data in Table 14 show that variants HSA-E492D+K573Pand HSA-E492G+K573P show higher affinity for shFcRn as compared to WTalbumin and the single K573P variant.

TABLE 15 Binding affinity of albumin variants (with two alterations) toshFcRn Binding % increase in SEQ ID response binding affinity AnalyteNo. (RU) compared to WT WT HSA 2 40.7 — HSA-K573P 3 55.3 35.9HSA-K574D + K573P 121 47.2 16.0 HSA-K574F + K573P 122 49.2 20.9HSA-K574G + K573P 123 52.1 28.0 HSA-K574H + K573P 124 53.4 31.2HSA-K574N + K573P 125 38.4 −5.7 HSA-K574S + K573P 126 40.8 0.2HSA-Q580K + K573P 128 53.7 31.9 HSA-Q580R + K573P 129 29.3 −28.0

The data in Table 15 show that introduction of substitutions to theK573P variant which had individually demonstrated an improvement inaffinity for shFcRn (Table 12), produced a range of binding responses,from a reduction in affinity as compared to WT albumin, through tovariants showing increased affinity.

TABLE 16 Binding affinity of albumin variants to shFcRn Fold SEQ Ka KdMean difference ID (10³/ (10⁻³/ KD KD from Analyte NO: Ms) s) (μM) (μM)WT HSA WT HSA 2 17.3 63.0 3.6 3.6 — HSA-K573P (run 1) 3 8.1 3.4 0.420.46 7.8 HSA-K573P (run 2) 3 8.0 4.0 0.5 HSA-E492H + 111 11.2 3.7 0.330.37 9.7 K573P (run 1) HSA-E492H + 111 11.4 4.7 0.41 K573P (run 2)HSA-E492R + 113 10.3 4.3 0.42 0.42 8.6 K573P HSA-K574G + 123 11.5 5.50.48 0.53 6.8 K573P (run 1) HSA-K574G + 123 11.7 6.8 0.58 K573P (run 2)HSA-K574H + 124 12.6 2.7 0.22 0.25 14.4 K573P (run 1) HSA-K574H + 12410.8 3.08 0.29 K573P (run 2) HSA-Q580K + 128 13.4 1.9 0.14 0.15 24.0K573P (run 1) HSA-Q580K + 128 13.2 2.19 0.16 K573P (run 2)

The data in Table 16 show that variants HSA-K574H+K573P andHSA-Q580K+K573P possess considerably higher affinity for shFcRn ascompared to WT albumin and the single K573P variant.

TABLE 17 Binding affinity of albumin variants to shFcRn Fold SEQ Ka KdMean difference ID (10³/ (10³/ KD KD from Analyte NO: MS) s) (μM) (μM)WT HSA WT HSA 2 9.7 72.2 7.4 6.9 — 8.9 57.3 6.4 HSA-K573P 3 6.1 3.9 0.640.7 9.9 5.4 4.1 0.76 HSA-D550K + 117 7.1 2.8 0.4 0.5 13.8 K573P 5.6 3.30.6 HSA-D550K + 130 10.1 36.6 3.6 3.75 1.8 K574H 8.8 34.8 3.9HSA-D550K + 132 12.9 22.6 1.7 1.6 4.3 Q580K 11.9 18.5 1.5 HSA-D550K +131 6.5 1.6 0.25 0.31 22.3 K574H + K573P 6.2 2.4 0.38 HSA-K574H + 1357.5 1.9 0.26 0.28 24.6 Q580K + K573P 6.0 1.8 0.3

The data in Table 17 show varying effects of the introduction ofmutations at positions 550, 573, 574 and 580. For example,HSA-K574H+K573P showed increased binding compared to WT albumin (Table16). Introduction of the K574H mutation into HSA-Q580K+K573P (Table 16)to produce HSA-K574H+Q580K+K573P resulted in no improvement in bindingaffinity (compared to WT albumin) over HSA-Q580K+K573P. In contrast,introduction of this mutation into HSA-D550K+K573P, producingHSA-D550K+K574H+K573P, has resulted in an increase in affinity comparedto WT albumin and HSA-D550K+K573P.

TABLE 18 Binding affinity of albumin variants to shFcRn Fold SEQ Ka KdMean difference ID (10³/ (10³/ KD KD from Analyte NO: Run Cycle MS) s)(μM) (μM) WT HSA WT HSA 2 1 1 11.1 68.1 6.1 7.10 — 2 1 11.2 89.3 8.0 212.5 90.8 7.2 HSA- 3 1 1 6.0 3.8 0.64 0.86 8.3 K573P 2 1 5.0 5.3 1.1 25.4 4.5 0.83 E492D + 114 1 1 7.8 1.3 0.16 0.18 39.4 K573P + 2 1 7.3 1.60.21 K574H + 2 7.9 1.3 0.16 Q580K E492G + 115 1 1 7.8 1.3 0.16 0.22 32.3K573P + 2 1 7.0 1.7 0.25 K574H + 2 5.3 1.3 0.24 Q580K

The data in Table 18 show that variants containing four substitutions atpositions 492 573P, 574 and 580 show a marked increase in bindingaffinity as compared to variants containing three mutations, such asHSA-K574H+Q580K+K573P (Table 17), variants containing two mutations,such as K574H+K573P and Q580K+K573P (Table 16) and the single K573Pvariant or WT albumin.

Example 3 Production of Further Combination Variants

Single or multiple further mutations were introduced into the templateplasmids listed in Table 19 to produce the indicated variants. Mutagenicforward primers and non-mutagenic reverse primers were used to introducethe desired changes, as listed in Table 20. The template plasmids weremethylated using the components described in Table 21, incubated at 37°C. for one hour and were then purified using a Qiagen QiaQuick PCRpurification kit according to manufacturer's instructions. Themethylated template was then used in mutagenic PCR reactions (Tables 22and 23) according to the oligonucleotide/template combination detailedin Table 19 to produce the required HSA variants. Following PCRreaction, 5 μl of each PCR reaction mixture was visualised by agarosegel electrophoresis to assess production of the plasmid and 5 μl ofplasmid was retained for further analysis.

TABLE 19 Variants and associated templates and oligonucleotides formutagenic PCR amplification Seq Oligo Oligo ID Variant forward reverseTemplate no. HSA E492D + D550K 01-f 01-r pDB4770 231 HSA E492D + K574H02-f 02-r pDB4770 232 HSA E492D + K573P + K574H 04-f 04-r pDB4770 233HSA E492D + K573P + Q580K 05-f 05-r pDB4770 234 HSA E492D + K573P +K574G + 06-f 06-r pDB4770 235 Q580K HSA E492D + K573P + K574H + 07-f07-r pDB4770 236 Q580R HSA E492F + K573P + K574H + 08-f 08-r pDB4771 237Q580K HSA E492F + K573P + K574G + 06-f 06-r pDB4771 238 Q580K HSAE492F + K573P + K574H + 07-f 07-r pDB4771 239 Q580R HSA E492G + D550K01-f 01-r pDB4772 240 HSA E492G + K574H 02-f 02-r pDB4772 241 HSAE492G + K573P + Q580K 05-f 05-r pDB4772 242 HSA E492G + K573P + K574G +06-f 06-r pDB4772 243 Q580K HSA E492G + K573P + K574H + 07-f 07-rpDB4772 244 Q580R HSA D550L + K574H 02-f 02-r pDB4791 245 HSA D550L +K573P + K574H 04-f 04-r pDB4791 246 HSA D550L + Q580K 03-f 03-r pDB4791247 HSA D550L + K573P + Q580K 05-f 05-r pDB4791 248 HSA D550M + K574H02-f 02-r pDB4792 249 HSA D550M + K573P + K574H 04-f 04-r pDB4792 250HSA D550M + Q580K 03-f 03-r pDB4792 251 HSA D550M + K573P + Q580K 05-f05-r pDB4792 252 HSA E492D + D550K + K573P 01-f 01-r pDB4990 253 HSAE492G + D550K + K573P 01-f 01-r pDB4992 254 HSA E492G + D550K + K574H29-f 29-r pDB5027 255 HSA E492D + D550K + K573P + 28-f 28-r pDB5028 256K574H HSA E492G + D550K + K573P + 29-f 29-r pDB5028 257 K574H HSAE492D + D550K + Q580K 28-f 28-r pDB5029 258 HSA E492G + D550K + Q580K29-f 29-r pDB5029 259 HSA E492D + D550K + K573P + 28-f 28-r pDB5030 260Q580K HSA E492G + D550K + K573P + 29-f 29-r pDB5030 261 Q580K HSAE492D + K574H + Q580K 28-f 28-r pDB5031 262 HSA E492G + K574H + Q580K29-f 29-r pDB5031 263 HSA E492D + K573P + K574H + 28-f 28-r pDB5032 264Q580K HSA D550K + K573P + K574H + 01-f 01-r pDB5032 265 Q580K HSAE492D + D550K + K573P + 01-f 01-r pDB5091 266 K574H + Q580K HSA E492G +D550K + K573P + 01-f 01-r pDB5092 267 K574H + Q580K (f = forward, r =reverse)

TABLE 20 Oligonucleotides for mutagenic PCR amplification. SEQ ID OligoSequence (5′ to 3′) No. 01-fGAACAATTGAAGGCTGTCATGGATAAGTTCGCTGCTTTCGTTGAAA 268 AG 01-rATCCATGACAGCCTTCAATTGTTCCTTAGTAGCCTT 269 02-fCTTGTTTCGCTGAAGAAGGTAAGCACTTGGTCGCTGCTTCCCAA 270 02-rCTTACCTTCTTCAGCGAAACAAGTTTCCTTATCATC 271 03-fTAAGAAGTTGGTCGCTGCTTCCAAGGCTGCCTTAGGTTTGTAATAA 272 03-rGGAAGCAGCGACCAACTTCTTACCTTCTTCAGC 273 04-fGAAACTTGTTTCGCTGAAGAAGGTCCACACTTGGTCGCTGCTTCCC 274 AA 04-rACCTTCTTCAGCGAAACAAGTTTCCTTATCATCAGC 275 05-fGAAACTTGTTTCGCTGAAGAAGGTCCAAAGTTGGTCGCTGCTTCCA 276 AGGCTGCCTTAGGTTTGTAA05-r ACCTTCTTCAGCGAAACAAGTTTCCTTATCATCAGC 277 06-fGAAACTTGTTTCGCTGAAGAAGGTCCAGGTTTGGTCGCTGCTTCCA 278 AGGCTGCCTTAGGTTTGTAA06-r ACCTTCTTCAGCGAAACAAGTTTCCTTATCATCAGC 279 07-fGAAACTTGTTTCGCTGAAGAAGGTCCACACTTGGTCGCTGCTTCCA 280 GAGCTGCCTTAGGTTTGTAA07-r ACCTTCTTCAGCGAAACAAGTTTCCTTATCATCAGC 281 08-fGAAACTTGTTTCGCTGAAGAAGGTCCACACTTGGTCGCTGCTTCCA 282 AGGCTGCCTTAGGTTTGTAA08-r ACCTTCTTCAGCGAAACAAGTTTCCTTATCATCAGC 283 28-fGAAGACCATGTTTCTCTGCTTTGGACGTCGACGAAACTTACGTTC 284 28-rCAAAGCAGAGAAACATGGTCTTCTGTTAACCAAAGA 285 29-fGAAGACCATGTTTCTCTGCTTTGGGTGTCGACGAAACTTACGTTC 286 29-rCAAAGCAGAGAAACATGGTCTTCTGTTAACCAAAGA 287 (f = forward, r = reverse)

TABLE 21 Methylation reaction components Template DNA 2.5 μg 10X Buffer(New England Biolabs) 5 μl Dam methylase (New England Biolabs) 1 μl Sadenosylmethionine (80 μM) (New England Biolabs) 12.5 μl H₂O Up to 50 μl

TABLE 22 PCR reaction components Template (5 ng/μl) 1 μl 5x buffer 10 μldNTP (10 mM) 1 μl Sterile water 35 Forward primer (20 μM) 1.25 μlReverse primer (20 μM) 1.25 μl Q5 polymerase 0.5 μl

TABLE 23 PCR reaction conditions for construction of variants listed inTable 19 Temperature Cycle Length Number of cycles 98° C. 2 min 1 98° C.10 sec 30 65° C.*/60° C.^(#) 30 sec 72° C. 5 min 72° C. 7 min 140 μl of each of the PCR-generated plasmids was prepared for in vivorecombination by the addition of 0.5 μl of each of DpnI, NsiI and PvuIrestriction enzymes (New England Biolabs), followed by incubation at 37°C. for one hour. 3 μl of the prepared plasmid, 3 μl ofAcc651/BamHI-digested pDB3936 and 1 μl of salmon sperm DNA were used totransform S. cerevisiae according to the protocol described inExample 1. The host strain used was a S. cerevisiae strain derived fromDYB7 ura3 (Payne et al 2008, Appl. Environ. Microbiol. 74(24):7759-7766) with two additional copies of PDI1 integrated into thegenome. Single colonies were patched onto BMMD+CSM-leu plates (asdescribed in WO 2012/150319, incorporated herein by reference) to enableassessment of expression prior to production of yeast stocks. Stockswere produced by the 48-hour method described in Example 1. 200 μl ofeach yeast strain was used to inoculate 10 ml BMMS in 50 ml shake flask,followed by incubation at 30° C., 200 rpm for four days. Culturesupernatant was harvested by centrifugation at 3000 rpm for 5 minutes.

Albumin variants were purified from 10 mL shake flasks using a singlechromatographic step with an albumin affinity matrix (AlbuPure™—ProMeticBioSciences, Inc.). Micro-scale affinity chromatography was performed onan automated platform (Perkin Elmer, Janus) with 200 μL custom packedAtoll columns with 8 run in parallel in a 96-well format using the sameprocedure as described in WO 2011/051489 (incorporated herein byreference), with volumes scaled down appropriately.

Final concentration of the samples was determined by Absorbance at 280nm using a UV microplate and a plate reader, readings were blankcorrected against PBS and the concentrations calculated based on anextinction coefficient of 0.52AU/cm for a 1 mg/mL solution (AU:absorbance units).

Example 4 Analysis of Further Combination Variants

Kinetic analyses using bio-layer interferometry were performed on anOctet Red-96 instrument (ForteBio). Immobilisation of GST-tagged shFcRn(shFcRn-GST/FLAG) was carried out on AR2G biosensors using ForteBioamine coupling chemistry following the instructions from themanufacturer. shFcRn-GST/FLAG-refers to GST-tag(glutathione-transferase) and a FLAG-tag (DYKDDDDK) on the C-terminal ofthe alpha chain of FcRn. Prior to use, the GST/FLAG-tagged shFcRn waspurified using IgG affinity chromatography. More specifically, theGST/FLAG-tagged shFcRn was captured on a GSTrap column and eluted withreduced glutathione. shFcRn was dialyzed into PBS into pH 7.4 andfurther purified using IgG Sepharose™ 6 Fast Flow (GE Healthcare). TheshFcRn was captured on the resin in 50 mM Na-acetate, 150 mM NaCl pH 5.5and eluted with PBS pH 7.4. The eluted shFcRn was concentrated to 2-5mg/mL and stored at −20° C. until use. Immobilised level ofshFcRn-GST/FLAG was at a response level more than 1 nm, and achievedusing a FcRn concentration of 2-10 μg/mL in sodium acetate, followed byethanolamine quenching of the amine coupling reaction. The sensors wereeither used directly or soaked in 15% (w/v) sucrose and dried until use.

Kinetic analyses were performed using micro-scale affinity purifiedalbumin variants diluted 2-, 4- and 8-fold in BMMD fermentation media(as described WO 2011/051489, incorporated herein by reference)supplemented with 100 mM sodium acetate and adjusted to pH 5.5.Association (120s) and dissociation (300s) were performed at 30° C. andshaking at 1000 rpm. ForteBio software was used for data evaluation andcalculation of KD values as well as association and dissociationconstants which were calculated using the HSA concentrations determinedby OD280 nm measurement.

TABLE 24 Binding affinity of albumin variants to shFcRn KD Fold SEQAver- difference ID age relative to No: Variant μM WT HSA 231 HSAE492D + D550K 0.12 1.2 232 HSA E492D + K574H 0.14 1.1 233 HSA E492D +K573P + K574H 0.04 3.8 234 HSA E492D + K573P + Q580K 0.03 5.8 235 HSAE492D + K573P + K574G + Q580K 0.04 3.9 236 HSA E492D + K573P + K574H +Q580R 0.03 5.5 237 HSA E492F + K573P + K574H + Q580K 0.03 6.0 238 HSAE492F + K573P + K574G + Q580K 0.06 2.6 239 HSA E492F + K573P + K574H +Q580R 0.03 4.5 240 HSA E492G + D550K 0.13 1.2 241 HSA E492G + K574H 0.141.1 242 HSA E492G + K573P + Q580K 0.03 5.8 243 HSA E492G + K573P +K574G + Q580K 0.04 3.9 244 HSA E492G + K573P + K574H + Q580R 0.03 5.5245 HSA D550L + K574H 0.06 2.4 246 HSA D550L + K573P + K574H 0.04 4.2247 HSA D550L + Q580K 0.10 1.5 248 HSA D550L + K573P + Q580K 0.03 5.9249 HSA D550M + K574H 0.13 1.2 250 HSA D550M + K573P + K574H 0.03 5.0251 HSA D550M + Q580K 0.10 1.5 252 HSA D550M + K573P + Q580K 0.02 7.2253 HSA E492D + D550K + K573P 0.03 4.8 254 HSA E492G + D550K + K573P0.03 4.5 255 HSA E492G + D550K + K574H 0.12 1.3 256 HSA E492D + D550K +K573P + K574H 0.03 4.6 258 HSA E492D + D550K + Q580K 0.09 1.7 259 HSAE492G + D550K + Q580K 0.08 1.8 260 HSA E492D + D550K + K573P + Q580K0.03 5.2 261 HSA E492G + D550K + K573P + Q580K 0.02 6.1 262 HSA E492D +K574H + Q580K 0.11 1.4 263 HSA E492G + K574H + Q580K 0.08 1.9 264 HSAE492D + K573P + K574H + Q580K 0.03 5.3 265 HSA D550K + K573P + K574H +Q580K 0.02 6.1 266 HSA E492D + D550K + K573P + K574H + 0.02 6.5 Q580K267 HSA E492G + D550K + K573P + K574H + 0.02 6.9 Q580K 2 WT HSA 0.15 1.0

Example 5 Further Analysis of Selected Combination Variants

A subset of the variants (Table 25) described in Table 19 were selectedfor further analysis. 3 μl of the PCR reactions were digested with 7 μlof a reaction mix containing 10 μl of buffer 4 (New England Biolabs), 5μl Dpnl and 55 μl of water. Reaction mixtures were incubated at 37° C.for 1.5 hours and were then purified using a Qiagen QiaQuick PCRpurification kit according to manufacturer's instructions. 2 μl of theprepared plasmids were used to transform competent E. coli 10-beta cells(New England Biolabs). Plasmid DNA was prepared utilising a Qiagen PlusMaxiprep kit, according to manufacturer's instructions. The resultingplasmids were sequenced to ensure the desired mutations had beenintroduced. Plasmid preparation and yeast transformations were performedas described in example 1, using strain BXP10 cir°. 24 hour yeast stockswere produced as described in example 1.

TABLE 25 Variant and plasmid number Con- SEQ Variant Plasmid struct IDNo HSA E492D + K574H pDB5386 HSAE-2 232 HSA E492D + K573P + Q580KpDB5387 HSAE-5 234 HSA D550L + K573P + K574H pDB5388 HSAE-19 246 HSAD550L + K573P + Q580K pDB5389 HSAE-21 248 HSA D550M + Q580K pDB5390HSAE-24 251 HSA E492D + D550K + K573P pDB5391 HSAE-26 253 HSA E492G +D550K + K573P pDB5392 HSAE-27 254 HSA E492G + D550K + K573P + pDB5393HSAE-31 257 K574HThe variants listed in Table 26 and 27 were analyzed by SPR using aBiacore 3000 instrument as described in Example 2 with the exceptionthat, prior to use, the His-tagged shFcRn was purified using IgGaffinity chromatography. More specifically, the His-tagged shFcRn wascaptured on a Ni-HiTrap column and eluted with imidazole. The shFcRn wascaptured on the resin in 50 mM Na-acetate, 150 mM NaCl pH 5.5 and elutedwith PBS pH 7.4. The eluted shFcRn was concentrated to 2-5 mg/mL andstored at −20° C. until use. using shFcRn-HIS

TABLE 26 SPR analysis of binding affinity of HSA variants to shFcRn FoldSEQ Ka Kd difference ID (10³/ (10⁻³/ KD relative to Variant No Ms) s) μMWT HSA HSA WT 2 10.2 76.3 7.5 — HSA K573P 3 11.2 7.1 0.63 11.9 HSAE492D + K574H 232 17.5 48.7 2.8 2.7 HSA E492D + K573P + 234 19.5 2.20.11 68.2 Q580K HSA D550L + K573P + 246 15.6 3.6 0.23 32.6 K574H HSAD550M + Q580K 251 21.1 28.2 1.3 5.8 HSA E492D + D550K + 253 17.6 3.20.18 41.7 K573P HSA E492G + D550K + 254 20.0 3.2 0.16 46.9 K573P HSAE492G + D550K + 257 17.0 2.6 0.15 50 K573P + K574H HSA D550L + K573P +248 18.0 1.7 0.09 83.3 Q580K

TABLE 27 SPR analysis of binding affinity of HSA variants to shFcRn FoldSEQ Ka Kd difference ID (10³/ (10⁻³/ KD relative to Variant No Ms) s) μMWT HSA HSA WT 2 16.7 101 6.1 — HSA K573P 3 16.4 9.75 0.6 10.2 HSAD550K + K573P + 131 27.5 4.4 0.16 38.1 K574H HSA K573P + K574H + 13524.7 3.2 0.13 46.9 Q580K HSA D550K + K573P + 133 44.2 1.9 0.05 122 Q580KHSA D550L + K573P + 248 36.3 2.12 0.06 101.7 Q580KThe data of Table 26 and 27 show an improvement in affinity over WT HSAin variants containing a combination of substitutions. Generally,variants containing three or four substitutions show improved bindingcharacteristics over those containing two substitutions. Generally,inclusion of Q580K contributes substantially to an improved bindingaffinity.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

What is claimed is:
 1. A polypeptide which is a variant of albumin,fragment thereof or fusion polypeptide comprising said variant albuminor a fragment thereof having an altered binding affinity to FcRncompared with the binding affinity of a parent albumin, referencealbumin, fragment thereof or fusion polypeptide comprising said parentalbumin, reference albumin or fragment or fusion thereof to FcRn,wherein the polypeptide comprises alterations at two or more positionsselected from positions corresponding to positions (a) 492 and 580; (b)492 and 574; (c) 492 and 550; (d) 550 and 573; (e) 550 and 574; (f) 550and 580 in SEQ ID NO:
 2. 2. A polypeptide which is a variant of albumin,fragment thereof or fusion polypeptide comprising said variant albuminor a fragment thereof having an altered binding affinity to FcRncompared with the binding affinity of a parent albumin, referencealbumin, fragment thereof or fusion polypeptide comprising said parentalbumin, reference albumin or fragment or fusion thereof to FcRn,wherein: (a) the polypeptide comprises alterations at two or morepositions, in which at a position corresponding to position 492 of SEQID NO: 2 there is an alteration to generate at a position correspondingto position an amino acid from the group consisting of A, C, D, F, H, I,K, L, M, N, P, Q, R, S, T, V, W, Y, preferably D and at a positioncorresponding to position 573 of SEQ ID NO: 2 there is an alteration togenerate at a position corresponding to position to an amino acid fromthe group consisting of C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W,Y, preferably Y, W or H, or (b) at a position corresponding to position492 of SEQ ID NO: 2 there is an alteration to generate G and at aposition corresponding to position 573 there is an alteration togenerate A or P and the polypeptide comprises an additional alterationat a position selected from the group consisting of 550, 574 and
 580. 3.A polypeptide which is a variant of albumin, fragment thereof or fusionpolypeptide comprising said variant albumin or a fragment thereof havingan altered binding affinity to FcRn compared with the binding affinityof a parent albumin, reference albumin, fragment thereof or fusionpolypeptide comprising said parent albumin, reference albumin orfragment or fusion thereof to FcRn, wherein: (a) the polypeptidecomprises alterations at two or more positions, in which at a positioncorresponding to position 573 of SEQ ID NO: 2 there is an alteration togenerate an amino acid from the group consisting of A, C, D, E, F, G, H,I, L, M, N, Q, R, S, T, V, W, Y, preferably Y, W or H and at a positioncorresponding to position 574 of SEQ ID NO: 2 there is an alteration togenerate an amino acid from the group consisting of A, C, D, E, F, G, H,I, L, M, P, Q, R, S, T, V, W, Y, H, D, F, G, N, S or Y, more preferablyH, D, F or G, most preferably H, or (b) at a position corresponding toposition 573 of SEQ ID NO: 2 there is a P and at a positioncorresponding to position 574 of SEQ ID NO: 2 there is an N and thepolypeptide comprises an additional alteration at a position selectedfrom the group consisting of 492, 550, and
 580. 4. A polypeptide whichis a variant of albumin, fragment thereof or fusion polypeptidecomprising said variant albumin or a fragment thereof having an alteredbinding affinity to FcRn compared with the binding affinity of a parentalbumin, reference albumin, fragment thereof or fusion polypeptidecomprising said parent albumin, reference albumin or fragment or fusionthereof to FcRn, wherein (a) the polypeptide comprises alterations attwo or more positions, in which at a position corresponding to position573 of SEQ ID NO: 2 there is an alteration to generate an amino acidfrom the group consisting of A, C, D, E, F, G, H, I, L, M, N, Q, R, S,T, V, W, Y, preferably Y, W or H and at a position corresponding toposition 580 of SEQ ID NO: 2 there is an alteration to generate an aminoacid from the group consisting of C, D, E, F, G, H, I, L, M, N, P, R, S,T, V, W, Y, or (b) at a position corresponding to position 573 of SEQ IDNO: 2 there is an alteration to generate a P and at a positioncorresponding to position 580 of SEQ ID NO: 2 there is an alteration togenerate a K and the polypeptide comprises an additional alteration at aposition selected from the group consisting of 492, 550, and
 574. 5. Apolypeptide which is a variant of albumin, fragment thereof or fusionpolypeptide comprising said variant albumin or a fragment thereof havingan altered binding affinity to FcRn compared with the binding affinityof a parent albumin, reference albumin, fragment thereof or fusionpolypeptide comprising said parent albumin, reference albumin orfragment or fusion thereof to FcRn, wherein: (a) the polypeptidecomprises alterations at two or more positions, in which at a positioncorresponding to position 574 of SEQ ID NO: 2 there is an alteration togenerate an amino acid from the group consisting of A, C, D, E, F, G, H,I, L, M, P, Q, R, S, T, V, W, Y, H, D, F, G, N, S or Y, more preferablyH, D, F or G, most preferably H and at a position corresponding toposition 580 of SEQ ID NO: 2 there is an alteration to generate at aposition corresponding to position to an amino acid from the groupconsisting of A, C, D, E, F, G, H, I, L, M, N, P, R, S, T, V, W, Y, or(b) at a position corresponding to position 574 of SEQ ID NO: 2 there isan alteration to generate an N and at a position corresponding toposition 580 of SEQ ID NO: 2 there is an alteration to generate a K andthe polypeptide comprises an additional alteration at a positionselected from the group consisting of 492, 550, and
 573. 6. Thepolypeptide according to claim 1, wherein the polypeptide comprisesalterations at three or more positions selected from positionscorresponding to positions 492, 550, 573, 574 and 580 in SEQ ID NO: 2.7. The polypeptide of claim 1 wherein the reference albumin is HSA (SEQID No: 2) or a fragment thereof, or a fusion polypeptide comprising HSAor a fragment thereof, most preferably SEQ ID NO:
 2. 8. The polypeptideaccording to claim 1 having a stronger binding affinity to FcRn and/orlonger plasma half-life than a parent albumin, reference albumin,fragment thereof or fusion polypeptide comprising said parent albumin,reference albumin or fragment or fusion thereof.
 9. The polypeptideaccording claim 1, wherein the sequence identity of the polypeptide toSEQ ID NO: 2 is more than 80%, preferably more than 90%, more preferredmore than 95%, more preferred more than 96%, even more preferred morethan 97%, more preferred more than 98% and most preferred more than 99%.10. A fusion polypeptide comprising a polypeptide according to claim 1and a fusion partner polypeptide selected from a therapeutic,prophylactic, diagnostic, imaging or other beneficial moiety.
 11. Amethod for preparing a polypeptide which is a variant of albumin,fragment thereof or fusion polypeptide comprising said variant albuminor fragment thereof having a binding affinity to FcRn which is alteredcompared to the binding affinity of a reference albumin, fragment orfusion thereof to FcRn, comprising: (a) Providing a nucleic acidencoding a parent albumin having at least 80% sequence identity to SEQID NO: 2; (b) Modifying the sequence of step (a), to encode apolypeptide which is a variant albumin, fragment thereof or fusionpolypeptide comprising said variant albumin or fragment thereofcomprising alterations at two or more positions corresponding topositions selected from 492, 550, 573, 574 and 580 in SEQ ID NO: 2,preferably as described in claim 1; (c) Introducing the modifiedsequence of step (b) in a suitable host cell; (d) Growing the cells in asuitable growth medium under condition leading to expression of thepolypeptide; and (e) Recovering the polypeptide from the growth medium;wherein the polypeptide has an altered binding affinity to FcRn and/oran altered plasma half-life compared with the half-life of a parentalbumin, reference albumin, fragment thereof or fusion polypeptidecomprising said parent albumin, reference albumin or fragment or fusionthereof.
 12. The method of claim 11 wherein the reference albumin is HSA(SEQ ID No: 2) or a fragment thereof, or a fusion polypeptide comprisingHSA or a fragment thereof, most preferably SEQ ID NO:
 2. 13. The methodaccording to claim 11, wherein the sequence identity of the polypeptideto SEQ ID NO: 2 is more than 80%, preferably more than 90%, morepreferred more than 95%, more preferred more than 96%, even morepreferred more than 97%, more preferred more than 98% and most preferredmore than 99%.
 14. A conjugate comprising a polypeptide according toclaim 1 and a conjugation partner.
 15. An associate comprising apolypeptide according to claim 1 or obtainable by a method according toclaim 11 and a therapeutic, prophylactic, diagnostic, imaging or otherbeneficial moiety.
 16. A nanoparticle or microparticle comprising apolypeptide according to claim
 1. 17. A composition comprising apolypeptide according to claim 1 wherein the binding affinity of thepolypeptide, fusion polypeptide, conjugate, associate or nanoparticle ormicroparticle to FcRn is stronger than the binding affinity of acomposition comprising the corresponding parent albumin, referencealbumin, fragment thereof or fusion polypeptide, conjugate, associate ornanoparticle or microparticle comprising said parent albumin, referencealbumin or fragment or fusion thereof to FcRn.
 18. A compositionaccording to claim 17 where the binding affinity of the polypeptide,fusion polypeptide, conjugate, associate or nanoparticle ormicroparticle to FcRn is stronger than the binding affinity of HSA toFcRn.
 19. A composition according to claim 17, wherein the bindingcoefficient of the variant of the polypeptide, fusion polypeptide,conjugate, associate or nanoparticle or microparticle to FcRn is lessthan 0.9×KD of HSA to FcRn, more preferred less than 0.5×KD of HSA toFcRn, more preferred less than 0.1×KD of HSA to FcRn, even morepreferred less than 0.05×KD of HSA to FcRn, even more preferred lessthan 0.02×KD of HSA to FcRn and most preferred less than 0.01×KD of HSAto FcRn.
 20. The composition according to claim 17, further comprising acompound comprising an antibody binding domain (ABD) and a therapeutic,prophylactic, diagnostic, imaging or other beneficial moiety.
 21. Thecomposition according to claim 17, comprising a pharmaceuticallyacceptable carrier or excipient.
 22. A method for altering the bindingaffinity to FcRn or half-life preferably in plasma, of a moleculecomprising: (a) where the molecule is a polypeptide, fusing orconjugating the molecule to a polypeptide according to claim; (b) wherethe molecule is not a polypeptide, conjugating the molecule to apolypeptide according to claim.
 23. A method according to claim 22wherein the molecule is a therapeutic, prophylactic, diagnostic, imagingor other beneficial moiety.
 24. A nucleic acid encoding the polypeptideor fusion polypeptide of claim
 1. 25. A vector comprising a nucleic acidaccording to claim
 24. 26. A host cell comprising a nucleic acidaccording to claim
 24. 27. A method of prophylaxis, treatment ordiagnosis comprising administering a polypeptide according to claim 1 toa subject.